Patent Publication Number: US-2020283773-A1

Title: Nucleolin-targeting aptamers and methods of using the same

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This patent application claims the benefit of priority of U.S. Provisional Patent Application No. 62/555,745, filed Sep. 8, 2017, which is incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support by the National Institutes of Health under Award Number CA159826. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     This application is being filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing entitled “2018-09-10_5667-00448_ST25.txt” created on Sep. 10, 2018 and is 101,860 bytes in size. The Sequence Listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety. 
     INTRODUCTION 
     The protein nucleolin plays a critical role in repair of DNA double-stranded breaks (DSB) (Goldstein et al, PNAS, 2013). Mechanistically, nucleolin functions as a histone chaperone at the DSB, escorting the histone proteins H2A and H2B away from the nucleosome at the DNA break. This nucleosome disruption is required for the recruitment of repair enzymes and the repair of the DNA breaks. Therefore, inhibition of nucleolin results in sensitization of cells to DNA damaging agents. Importantly, the majority of human tumors overexpress nucleolin on the cell surface relative to normal cells, thus making nucleolin a tumor-preferential target. A nucleolin inhibitor would have the unique ability to specifically sensitize only tumor cells to DNA damaging agents as it should only target and internalize into cancerous cells. 
     Aptamers, small artificial RNA or DNA oligonucleotide ligands, can be selected to inhibit protein function and are also emerging as important tumor-targeting molecules. Additionally, they have many advantages over traditional antibody targeting agents, including ease of synthesis and amenability to chemical modification (Keefe et al, Nat Rev Drug Discov, 2010). Moreover, they exhibit antibody-like target affinities and specificities at a fraction of the size, allowing more efficient tumor penetration while maintaining the ability to discriminate between proteins that differ by only a few amino acids (reviewed in Conrad et al, Methods Enzymol, 1996; Obsorne et al, Chem Rev, 1997). 
     There is a need in the art for new aptamers that may bind to and/or inhibit the nucleolin protein. Such aptamers may be useful not only as new cancer treatments but also may facilitate the delivery of agents to the nucleus of a cell. 
     SUMMARY 
     In one aspect of the present invention, aptamers are provided. The aptamer may include a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 1-490, 494-515, or any one of the sequences described in the Tables or Figures disclosed herein (for example, Tables 1-4, 6-8 or  FIG. 11A-11B, 12A-12B, 13A-13C, 14A-14D, 15A-15B, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 ,  27 ,  28 A- 28 B,  29 ,  30 ,  31 ,  32 ,  33 ,  34 ,  35 ,  36  or  37 A- 37 B). In another aspect, the present invention relates to dimers, trimers, and tetramers including any one of the aptamers described herein. 
     In a further aspect of the present invention, pharmaceutical compositions including any of the aptamers described herein are provided. The pharmaceutical compositions may include a pharmaceutical carrier, excipient, or diluent. 
     In a still further aspect, the present invention relates to methods for treating cancer in a subject. The methods may include administering to the subject a therapeutically effective amount of any one of the aptamers, dimers, trimers, tetramers, or pharmaceutical compositions described herein. 
     In a still further aspect, methods of labeling or inhibiting nucleolin are provided. The methods include contacting nucleolin with any one of the compositions described herein to allow binding and possibly inhibition of the activity of the nucleolin. This contacting can be in vitro by adding the nucleolin to cells or may be in vivo by administering the compositions described herein to a subject. The compositions and aptamers provided herein are capable of binding to and possibly inhibiting the function of nucleolin. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the work flow demonstrating the selection of aptamer families capable of relocating into the nucleus after binding to nucleolin on cell surface. A random 2′Fluoro-pyrimidine RNA pool of sequences GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 491)-N 40 -CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492) (where N 40  represents 40 random nucleotides) was incubated for 20 min at 37° C. with nucleolin protein (in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl 2  and 0.01% bovine serum albumin) at ratios of RNA:protein varying from 187:1 to 133:1. RNA bound to protein was isolated by filtration through a 0.45 μm nitrocellulose membrane before RNA extraction, reverse transcription, PCR amplification and transcription to complete 1 round of selection. Each subsequent round of selection used the RNA pool transcribed from the previous round of selection, for a total of 7 rounds of SELEX against the nucleolin protein. The Round 6 RNA pool was also used to perform 2 Cell-SELEX rounds against both MCF-7 and Panc-1 cells. For the Cell-SELEX rounds, the Round 6 RNA pool was incubated with either MCF-7 or Panc-1 cells for 2 hrs at 37° C./5% CO 2  before using a high salt wash to remove non-internalized RNA. Cells were then tryspinized, washed again with high salt, and RNA extracted from the cell nuclei using the Invitrogen™ PARIS™ kit. RNA pools from Rounds 3, 5, 7 and 9 (Panc-1) were reverse transcribed, PCR amplified and analyzed by High-Throughput Sequencing. 
         FIGS. 2A-2D  show binding of the SELEX and Cell-SELEX rounds to the nucleolin protein (NCL). RNA pools from SELEX rounds 3, 6, and 7 or from Cell-SELEX Rounds 7-8 MCF-7 or Rounds 7-8 Panc-1 were end-labeled with  32 P. Nucleolin protein was serially diluted in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl 2  and 0.01% bovine serum albumin and incubated with a trace amount of the  32 P-labeled RNA pools. After incubation at 37° C., unbound RNA was captured on a nylon membrane and RNA-nucleolin complexes captured on a nitrocellulose membrane. The fraction of protein-bound RNA was determined via phosphorimaging of the nitrocellulose and nylon membranes. 
         FIGS. 3A-3B  show nucleolin-specific RNA aptamers bind to the RBD domain of nucleolin. ( FIG. 3A ) Map of truncated nucleolin mutants. From Chen et al. 2011 , JBC . ( FIG. 3B ) Southwestern blot showing the binding of the initial RNA aptamer library (Sell) versus SELEX round 6 (R6 NCL) to truncated nucleolin mutants expressed in MCF7 cells. 
         FIG. 4  shows binding analysis of the nucleolin (NCL) aptamers identified through high throughput sequencing. Aptamers were end-labeled with  32 P. Nucleolin protein was serially diluted in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl 2  and 0.01% bovine serum albumin and incubated with a trace amount of the  32 P-labeled RNA pools. After incubation at 37° C., unbound RNA was captured on a nylon membrane and RNA-nucleolin complexes captured on a nitrocellulose membrane. The fraction of protein-bound RNA was determined via phosphorimaging of the nitrocellulose and nylon membranes. 
         FIGS. 5A-5F  show binding of nucleolin aptamer truncates to the nucleolin protein. Aptamers were end-labeled with  32 P. Nucleolin protein was serially diluted in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl 2  and 0.01% bovine serum albumin and incubated with a trace amount of the  32 P-labeled RNA pools. After incubation at 37° C., unbound RNA was captured on a nylon membrane and RNA-nucleolin complexes captured on a nitrocellulose membrane. The fraction of protein-bound RNA was determined via phosphorimaging of the nitrocellulose and nylon membranes. 
         FIGS. 6A-6B  show nucleolin specific RNA aptamer EV3 sensitizes colon cancer cells to ionizing radiation. HCT 116 p53 -/- colon cancer cells were treated with 5 μg of indicated aptamers and exposed to 2Gy IR 48 h later. Cells were cultivated for 10 d and survival was assessed by MTT assay. 
         FIG. 7  shows EV3 does not sensitize HFF (human foreskin fibroblasts), that do not express nucleolin on cell surface, to radiation. hTERT-immortalized HFF cells that do not express nucleolin on cell surface were treated with 5 μg of indicated aptamers and exposed to 2Gy IR 48 h later. Cells were cultivated for 10 d and survival was assessed by MTT assay. 
         FIG. 8  shows EV3 and EV5 bind to nucleolin expressed on the cell surface in a concentration dependent manner. Flow cytometry analysis of MFI (mean fluorescence intensity) of DL650-labeled EV3 and EV5 after incubation of HCT116 p53-/- cells with the indicated aptamer concentrations. 
         FIGS. 9A-9D  show binding of Ev3 aptamer truncates to the nucleolin protein. Aptamers were end-labeled with  32 P. Nucleolin protein was serially diluted in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl 2  and 0.01% bovine serum albumin and incubated with a trace amount of the  32 P-labeled RNA pools. After incubation at 37° C., unbound RNA was captured on a nylon membrane and RNA-nucleolin complexes captured on a nitrocellulose membrane. The fraction of protein-bound RNA was determined via phosphorimaging of the nitrocellulose and nylon membranes. 
         FIG. 10  shows truncation of EV3 resulted in reduced activity as radiosensitizer. HCT 116 p53 -/- colon cancer cells were treated with 5 μg of indicated full-length aptamers or EV3 truncates and exposed to 2Gy IR 48 h later. Cells were cultivated for 10 d and survival was assessed by MTT assay. 
         FIGS. 11A-11B  show predicted secondary structures for a representative Family B aptamer (SEQ ID NO: 8). 
         FIGS. 12A-12B  show predicted secondary structures for a representative Family C aptamer (SEQ ID NO: 9). 
         FIGS. 13A-13C  show predicted secondary structures for a representative Family D aptamer (SEQ ID NO: 10). 
         FIGS. 14A-14D  show predicted secondary structures for a representative Family E aptamer (SEQ ID NO: 11). 
         FIGS. 15A-15B  show predicted secondary structures for a representative Family F aptamer (SEQ ID NO: 12). 
         FIG. 16  shows predicted secondary structures for Ev3min2 truncate aptamer (SEQ ID NO: 497). 
         FIG. 17  shows predicted secondary structures for Ev3min3 truncate aptamer (SEQ ID NO: 498). 
         FIG. 18  shows predicted secondary structures for Ev3min4 truncate aptamer (SEQ ID NO: 499). 
         FIG. 19  shows predicted secondary structures for Ev3min5 truncate aptamer (SEQ ID NO: 500). 
         FIG. 20  shows predicted secondary structures for Ev3min6 truncate aptamer (SEQ ID NO: 501). 
         FIG. 21  shows predicted secondary structures for Ev3min7 truncate aptamer (SEQ ID NO: 502). 
         FIG. 22  shows predicted secondary structures for Ev3min8 truncate aptamer (SEQ ID NO: 503). 
         FIG. 23  shows predicted secondary structures for Ev3min9 truncate aptamer (SEQ ID NO: 504). 
         FIG. 24  shows predicted secondary structures for Ev3min10 truncate aptamer (SEQ ID NO: 505). 
         FIG. 25  shows predicted secondary structures for Ev3min11 truncate aptamer (SEQ ID NO: 506). 
         FIG. 26  shows predicted secondary structures for Ev3min12 truncate aptamer (SEQ ID NO: 507). 
         FIG. 27  shows predicted secondary structures for Ev3min13 truncate aptamer (SEQ ID NO: 508). 
         FIGS. 28A-28B  show predicted secondary structures for Ev3min14 truncate aptamer (SEQ ID NO: 509) and Ev3min15 truncate aptamer (SEQ ID NO: 510). 
         FIG. 29  shows predicted secondary structures for Ev3min16 truncate aptamer (SEQ ID NO: 511). 
         FIG. 30  shows predicted secondary structures for Ev3min17 truncate aptamer (SEQ ID NO: 512). 
         FIG. 31  shows predicted secondary structures for Ev3min18 truncate aptamer (SEQ ID NO: 513). 
         FIG. 32  shows predicted secondary structures for Ev3min19 truncate aptamer (SEQ ID NO: 514). 
         FIG. 33  shows predicted secondary structures for Ev3min20 truncate aptamer (SEQ ID NO: 515). 
         FIG. 34  shows predicted secondary structures for Ev3min21 truncate aptamer (SEQ ID NO: 486). 
         FIG. 35  shows predicted secondary structures for Ev3min22 truncate aptamer (SEQ ID NO: 487). 
         FIG. 36  shows predicted secondary structures for Ev3min23 truncate aptamer (SEQ ID NO: 488). 
         FIGS. 37A-37B  show predicted secondary structures for Ev3min24 truncate aptamer (SEQ ID NO: 489) and Ev3min25 truncate aptamer (SEQ ID NO: 490). 
     
    
    
     DETAILED DESCRIPTION 
     Here, in the non-limiting Examples, the present inventors disclose new aptamers that may bind to and/or inhibit the nucleolin protein. The present inventors demonstrate that such aptamers may be useful not only to sensitize cancer cells to cancer treatments including, for example, ionizing radiation and chemotherapeutic agents, but also may facilitate the delivery of agents to the nucleus of a cell. 
     In one aspect of the present invention, aptamers are provided. As used herein, the term “aptamer” refers to single-stranded oligonucleotides that bind specifically to target molecules with high affinity. Aptamers can be generated against target molecules, such as nucleolin, by screening combinatorial oligonucleotide libraries for high affinity binding to the target (See, e.g., Ellington, Nature 1990; 346: 8 18-22 (1990), Tuerk, Science 249:505-1 0 (1990)). The aptamers disclosed herein may be synthesized using methods well-known in the art. For example, the disclosed aptamers may be synthesized using standard oligonucleotide synthesis technology employed by various commercial vendors including, without limitation, Integrated DNA Technologies, Inc. (IDT), Sigma-Aldrich, Life Technologies, or Bio-Synthesis, Inc. 
     The aptamer may include a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 1-490, 494-515, or any one of the sequences described in the Tables or Figures disclosed herein (for example, Tables 1-4, 6-8 or  FIG. 11A-11B, 12A-12B, 13A-13C, 14A-14D, 15A-15B, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 ,  27 ,  28 A- 28 B,  29 ,  30 ,  31 ,  32 ,  33 ,  34 ,  35 ,  36  or  37 A- 37 B). The aptamers described herein (i.e., SEQ ID NOS: 1-490, 494-515) may or may not include a 5′ constant region (GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 491)) that may be used, for example, to transcribe or purify the aptamers in vitro. The aptamers described herein (i.e., SEQ ID NOS: 1-490, 494-515) may or may not include a 3′ constant region (CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492)) that may be used, for example, to transcribe or purify the aptamers in vitro. In some embodiments, the aptamer may include a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the polynucleotide sequence-5′-GGGAGAGAGGAAGAGGGAUGGG (SEQ ID NO: 491)-A Variable Region-CAUAACCCAGAGGUCGAUAGUACUGGAUCCCCCC (SEQ ID NO: 492)-3′, wherein the variable region may include any one of SEQ ID NOS: 13-473 or a portion thereof. The portion of the indicated aptamers should be capable of binding to nucleolin. In some embodiments, the aptamer may include a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 480 (Ev3 Aptamer). 
     The terms “polynucleotide,” “nucleotide sequence,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases may refer to DNA or RNA of genomic, natural, or synthetic origin. 
     Regarding polynucleotide sequences, the terms “sequence identity,” “percent identity,” and “% identity” refer to the percentage of base matches between at least two nucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Sequence identity for a nucleotide sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known nucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. 
     Regarding polynucleotide sequences, sequence identity is measured over the length of an entire defined nucleotide sequence, for example, as defined by a particular sequence identified herein. Furthermore, sequence identity, as measured herein, is based on the identity of the nucleotide base in the nucleotide sequence, irrespective of any further modifications to the nucleotide sequence. For example, the polynucleotide nucleotide sequences described herein may include modifications to the nucleotide sequences such 2′flouro, 2′O-methyl, and inverted deoxythymidine (idT) modifications. These modifications are not considered in determining sequence identity. Thus if a base, for example, is a 2′fluoro adenine (or 2′O-methyl, etc.), it is understood to be an adenine for purposes of determining sequence identity with another sequence. Likewise, 3′ idT modifications to the polynucleotide sequences described herein also should not be considered in determining sequence identity. 
     Based on the general aptamer structure presented, for example, in  FIG. 11A-11B, 12A-12B, 13A-13C, 14A-14D, 15A-15B, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 ,  27 ,  28 A- 28 B,  29 ,  30 ,  31 ,  32 ,  33 ,  34 ,  35 ,  36  or  37 A- 37 B, a person of ordinary skill in the art would readily recognize that several modifications could be made to the sequence while preserving the overall structure and presumably the function of the aptamer. For example, in  FIG. 11A , a person of ordinary skill in the art could simply switch the first stem forming region GGGA and the tenth stem forming region UCCC to CCCU and AGGG, respectively, and still retain the stem structure of the aptamer. Additionally, modifications to the stem regions could be made that change the bases within the stem region but conserve the overall pyrimidine and purine base composition so that the stem region hybridizes at a similar melting temperature. A person of ordinary skill would also recognize that changes made to the aptamer that disturbed the general aptamer stem loop structure would likely result in an aptamer incapable of efficiently binding its target. 
     In some embodiments, the aptamer may have a dissociation constant (K D ) for the nucleolin protein that is less than 1000, 800, 600, 500, 450, 350, 250, 150, 125, 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 2.5, 2, 1, 0.5, or 0.1 nanomolar (nM). The K D  of an aptamer may be measured using the methodology used by the inventors in the Examples. 
     The aptamers may include a polynucleotide (RNA, DNA, or peptide nucleic acid (PNA)) that is in an unmodified form or may be in a modified form including at least one nucleotide base modification. Nucleotide base modifications of polynucleotides to, for example, protect the polynucleotide from nuclease degradation and/or increase the stability of the polynucleotide and are well-known in the art. Common nucleotide base modifications that may be used in accordance with the present invention include, without limitation, deoxyribonucleotides, 2′-O-Methyl bases, 2′-Fluoro bases, 2′ Amino bases, inverted deoxythymidine bases, 5′ modifications, and 3′ modifications. In some embodiments, the aptamer may include a polynucleotide including a modified form including at least one nucleotide base modification selected from the group consisting of a 2′fluoro modification, a 2′O-methyl modification, a 5′ modification, and a 3′ modification. 
     Typical 5′ modifications may include, without limitation, inverted deoxythymidine bases, addition of a linker sequence such as C6, addition of a cholesterol, addition of a reactive linker sequence which could be conjugated to another moiety such as a PEG. Typical 3′ modifications may include, without limitation, inverted deoxythymidine bases, and inverted abasic residues. 
     As additional 5′ and/or 3′ modifications, the aptamer may include a polynucleotide including a 5′ linker and/or a 3′ linker. Common 5′ and/or 3′ linkers for polynucleotides are known in the art and may include peptides, amino acids, nucleic acids, as well as homofunctional linkers or heterofunctional linkers. Particularly useful conjugation reagents that can facilitate formation of a covalent bond with an aptamer may comprise an N-hydroxysuccinimide (NHS) ester and/or a maleimide or using click chemistry. Typical 5′ and/or 3′ linkers for polynucleotides may include without limitation, amino C3, C4, C5, C6, or C12-linkers. 
     The aptamer may further include an agent. Suitable agents may include, without limitation, stability agents, detectable agents such as reporter moieties, and/or therapeutic agents. 
     As used herein, a “stability agent” refers to any substance(s) that may increase the stability and/or increase the circulation time of a polynucleotide in vivo. Typical stability agents are known in the art and may include, without limitation, polyethylene glycol (PEG), cholesterol, albumin, or Elastin-like polypeptide. 
     As used herein, a “detectable agent” refers to any substance(s) that may be detected using appropriate equipment. Suitable detectable agents may be, without limitation, a fluorophore moiety, an enzyme moiety, an optical moiety, a magnetic moiety, a radiolabel moiety, an X-ray moiety, an ultrasound imaging moiety, a photoacoustic imaging moiety, a nanoparticle-based moiety, or a combination of two or more of the listed moieties. 
     A “fluorophore moiety” may include any molecule capable of generating a fluorescent signal. Various fluorophore moieties are well-known in the art and/or commercially available. Exemplary fluorophore moieties include, without limitation, fluorescein, FITC, Alexa Fluor 488, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 750, and Alexa Fluor 790 (Life Technologies); Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7 (GE Healthcare); DyLight 350, DyLight 488, DyLight 594, DyLight 650, DyLight 680, DyLight 755 (Life Technologies); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); VivoTag680, VivoTag-S680, and VivoTag-S750 (PerkinElmer). 
     An “enzyme moiety” refers to polypeptides that catalyze the production of a detectable signal. Exemplary enzyme moieties may include, without limitation, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, or β-galactosidase. 
     “Optical moieties” may include, for example, any agents that may be used to produce contrast or signal using optical imaging such as luminescence or acousto-optical moieties. 
     “Magnetic moieties” may include, for example, a chelating agent for magnetic resonance agents. Chelators for magnetic resonance agents can be selected to form stable complexes with paramagnetic metal ions, such as Gd(III), Dy(III), Fe(III), and Mn(II). 
     Other exemplary detectable agents may include radiolabel moieties. Exemplary radioactive labels may include, without limitation,  99 Mo,  99m Tc,  64 Cu,  67 Ga,  186 Re,  188 Re,  153 Sm,  177 Lu,  67 Cu,  123 I,  124 I,  125 I n,  X 3N  15 O, and  18 F. 
     “X-ray moieties” may include, for example, any agents that may be used to produce contrast or signal using X-ray imaging such as iodinated organic molecules or chelates of heavy metal ions. 
     “Photoacoustic imaging moieties” may include photoacoustic imaging-compatible agents such as methylene blue, single-walled carbon nanotubes (SWNTs), and gold nanoparticles. Ultrasound imaging moieties may include, for example, any agents that may be used to produce contrast or signal using ultrasound imaging such as Levovist, Albunex, or Echovist. 
     A detectable agent may also be a nanoparticle-based moiety. A nanoparticle-based moiety is a nanoparticle that is capable of generating a signal. For example, silicon containing nanoparticles may be used to produce fluoresecence, luminescence, or another type of signal. Other exemplary nanoparticle-based moieties include, without limitation, nanospheres such as Kodak X-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Fisher Scientific); metal oxide nanoparticles; and quantum dots such as EviTags (Evident Technologies) or Qdot probes (Life Technologies). 
     As used herein, a “therapeutic agent” may be any substance that provides a therapeutic functionality when conjugated to any one of the aptamers described herein. Suitable therapeutic agents may include, without limitation, cytotoxic compounds, and particularly those shown to be effective in other drug conjugates. As used herein, a “cytotoxic compound” refers to any substance that disrupts the functioning of cells and/or causes the death of cells. Various therapeutic cytotoxic compounds are known in the art and may include, without limitation, DNA damaging agents, anti-metabolites, natural products and their analogs. Exemplary classes of cytotoxic compounds include enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, tubulin inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, auristatins, maytansinoids, differentiation inducers, and taxols. More specifically, suitable cytoxic compounds may include 5-fluorouracil, aclacinomycin, activated cytoxan, bisantrene, bleomycin, carmofur, CCNU, cis-platinum, daunorubicin, doxorubicin, DTIC, melphalan, methotrexate, mithromycin, mitomycin, mitomycin C, peplomycin pipobroman, plicamycin, procarbazine, retinoic acid, tamoxifen, taxol, tegafur, VP16, VM25, diphtheria toxin, botulinum toxin, geldanamycin, maytansinoids (including DM1), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and their analogues. Exemplary cytotoxic compounds may also include therapeutic radiopharmaceuticals including, without limitation,  186 Re,  188 Re,  153 Sm,  67 Cu,  105 Rh,  m Ag, and  192 Ir. 
     The aptamer and agent may be “linked” either covalently or non-covalently. Additionally, the aptamer and agent may be linked using the 5′ and/or 3′ linkers described herein. The aptamer and agent may be linked at the 5′ end and/or the 3′ end of the aptamer. To link the aptamer and agent non-covalently, the aptamer and the agent may be linked by a tag system. A “tag system” may include any group of agents capable of binding one another with a high affinity. Several tag systems are well-known in the art and include, without limitation, biotin/avidin, biotin/streptavidin, biotin/NeutrAvidin, or digoxigenin (DIG) systems. In some embodiments, the tag system comprises biotin/avidin or biotin/streptavidin. In such embodiments, the aptamer may be modified at either the 5′ or 3′ end to include biotin while the agent may be modified to include streptavidin or avidin. Alternatively, the aptamer may be modified at either the 5′ or 3′ end to include streptavidin or avidin while the agent may be modified to include biotin. 
     In another aspect, the present invention relates to dimers, trimers, and tetramers including any one of the aptamers described herein. A “dimer” refers to the linking together of two aptamer molecules in order to, for example, to increase the stability and/or increase the circulation time of a polynucleotide in vivo. A “trimer” refers to the linking together of three aptamer molecules in order to, for example, to increase the stability and/or increase the circulation time of a polynucleotide in vivo. A “tetramer” refers to the linking together of four aptamer molecules in order to, for example, to increase the stability and/or increase the circulation time of a polynucleotide in vivo. The aptamer molecules may be linked together covalently, noncovalently, or a combination of both. The aptamer molecules may be linked at their 5′ or 3′ ends. To link the aptamers noncovalently, the aptamers may be linked by a tag system or through a scaffold system. 
     In a further aspect of the present invention, pharmaceutical compositions including any of the aptamers described herein are provided. The pharmaceutical compositions may include a pharmaceutical carrier, excipient, or diluent (i.e., agents), which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often a pharmaceutical composition may include an aqueous pH buffered solution. Examples of pharmaceutical carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ brand surfactant, polyethylene glycol (PEG), and PLURONICS™ surfactant. In some embodiments, the pharmaceutical carrier may include a buffer including about 20 mM Hepes, pH 7.4; 150 mM NaCl; 1 mM CaCl 2 ; 1 mM MgCl 2 ; 5 mM KCl. 
     In a still further aspect, the present invention relates to methods for treating cancer in a subject. The methods may include administering to the subject a therapeutically effective amount of any one of the aptamers, dimers, trimers, tetramers, or pharmaceutical compositions described herein. The subject may be any mammal, suitably a human, domesticated animal such as a dog or cat, or a mouse or rat. Optionally, the present methods may further include administering a chemotherapeutic agent or radiation therapy to the subject. 
     Exemplary cancers in accordance with the present invention include, without limitation, colon, primary and metastatic breast, ovarian, liver, pancreatic, prostate, bladder, lung, osteosarcoma, pancreatic, gastric, esophageal, skin cancers (basal and squamous carcinoma; melanoma), testicular, colorectal, urothelial, renal cell, hepatocellular, leukemia, lymphoma, multiple myeloma, head and neck, and central nervous system cancers or pre-cancers. 
     Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in the subject, reducing progression of a cancer to a more aggressive form, reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or at risk of developing the disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc. 
     Optionally, the present methods may further include administering a chemotherapeutic agent and/or radiation therapy to the subject. Without being limited by theory, the present inventors conjecture (and demonstrate in the Examples) that aptamers that block nucleolin function in cancer cells can sensitize cancer cells to DNA-damaging agents such as chemotherapeutic agents and radiation therapy. In some embodiments, the aptamer-containing composition described herein is administered prior to, simultaneously with, or after the chemotherapeutic agent and/or radiation therapy. In some embodiments, the aptamer-containing composition is administered prior to the administration of the optional chemotherapeutic agent and/or radiation therapy. 
     Chemotherapeutic agents are compounds that may be used to treat cancer. Suitable chemotherapy agents may include, without limitation, 5-fluorouracil, aclacinomycin, activated cytoxan, bisantrene, bleomycin, carmofur, CCNU, cis-platinum, daunorubicin, doxorubicin, DTIC, melphalan, methotrexate, mithromycin, mitomycin, mitomycin C, peplomycin pipobroman, plicamycin, procarbazine, retinoic acid, tamoxifen, taxol, tegafur, VP16, or VM25. 
     In some embodiments, the chemotherapeutic agent may be a DNA-damaging agent including, without limitation, cisplatin, carboplatin, picoplatin, oxaliplatin, methotrexate, doxorubicin, or daunorubicin, 5-fluorouracil, capecitabine, floxuridine, and gemcitabine, and the purine analogs 6-mercaptopurine, 8-azaguanine, fludarabine, and cladribine. The optional radiation therapy in the present methods may include one or more doses of between 1 Gy and 30 Gy. Suitably, the radiation therapy includes a single fraction dose of 12, 15, 18, 20, 21, 23, 25, or 28 Gy. 
     The chemotherapeutic agent and/or radiation therapy may be administered in any order in relation to the aptamer-containing compositions described herein, at the same time or as part of a unitary composition. The aptamer-containing composition and chemotherapeutic agent and/or radiation therapy may be administered such that one composition or therapy is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more. 
     An “effective amount” or a “therapeutically effective amount” as used herein means the amount of a composition that, when administered to a subject for treating a state, disorder or condition is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the composition, formulation or combination, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated. 
     The compositions (i.e., those including the aptamers described herein) described herein may be administered by any means known to those skilled in the art, including, but not limited to, intratumoral, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, or transmucosal absorption. Thus the compositions may be formulated as an ingestable, injectable, topical or suppository formulation. Within broad limits, administration of larger quantities of the aptamer-containing compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen. 
     It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the aptamer-containing compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compositions or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations. 
     The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual prophylactic or treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the compound will reduce symptoms of the condition at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to pre-treatment symptoms or symptoms is left untreated. It is specifically contemplated that pharmaceutical preparations and compositions may palliate or alleviate symptoms of the disease without providing a cure, or, in some embodiments, may be used to cure the disease or disorder. 
     The effectiveness of the aptamer-containing composition in treating the cancer or reducing the likelihood of resistance can be measured by tracking the growth of the tumor or the growth rate of the tumor or cancer cells. A decrease in tumor size or in the rate of tumor growth is indicative of treatment of the cancer. 
     The aptamers disclosed herein may also be used in methods of labeling or inhibiting nucleolin. As disclosed herein the aptamers provided bind to nucleolin and may be used to inhibit nucleolin. In some instances the aptamers are trafficked with the nucleolin to the nucleus of the cell when the aptamer is contacts the cell. The aptamers may be combined with an agent as described above and if the agent is a reporter moiety the agent may allow nucleolin to be labeled within the cell or to bring the agent in contact with nucleolin. Nucleolin may be contacted with the aptamer directly or indirectly in vivo, in vitro, or ex vivo. Contacting encompasses administration to a cell, a culture of cells, tissue, mammal, patient, or human expressing nucleolin. Further, contacting a cell includes adding an agent to a cell culture. Other suitable methods may include introducing or administering an agent to a cell, tissue, mammal, or patient using appropriate procedures and routes of administration as defined above. 
     The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements. 
     Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise. 
     No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference in their entirety, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references. 
     Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a protein” or “an RNA” should be interpreted to mean “one or more proteins” or “one or more RNAs,” respectively. 
     The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. 
     EXAMPLES 
     Example 1—Development of Nucleolin-Binding Aptamers 
     With the goal of developing an aptamer that binds and/or inhibits the nucleolin protein, we performed a dual protein and cell selection via systematic evolution of ligands by exponential enrichment (SELEX) using a modified RNA library ( FIG. 1 ). First, SELEX was performed against a recombinant nucleolin protein (SEQ ID NO: 493) resulting in an RNA library enriched in clones specific for nucleolin after 6 rounds of selection ( FIG. 2A ). As a 7 th  round of SELEX did not improve the aptamer pool&#39;s affinity for the nucleolin protein ( FIG. 2B ), we moved forward with the pool of RNA from the 6 th  round of SELEX (R6 NCL). To identify nucleolin-specific RNAs capable of binding to nucleolin on cell surface and subsequently transporting to the nucleus, the R6 NCL RNA pool was incubated with either MCF-7 or Panc-1 cells ( FIG. 1 ). The nuclei were then isolated and the aptamer pool that reached this compartment was amplified. After 2 rounds of cellular selection with either MCF-7 or Panc-1 cells, the RNA library was further enriched for aptamers capable of binding to the nucleolin protein ( FIGS. 2C &amp; 2D ). 
     We previously demonstrated that nucleolin interacts with Rad50, a member of the MRN complex, through its C-terminal RGG domain and that this interaction is essential for recruitment of nucleolin to the DNA damage site and repair of the DSB (Goldstein et al. 2013, PNAS). Thus, we estimated that in order to achieve a disruption of the nucleolin-Rad50 interaction and the inhibition of DSB repair required for radiosensitization, our nucleolin aptamer would need to bind to either the RGG domain itself or to the RBD domain in the proximity of the C-terminus. In fact, we found that the R6 NCL RNA aptamer pool binds to the RBD domain ( FIGS. 3A-3B ), suggesting that these aptamers may be able to inhibit the nucleolin-Rad50 interaction that is crucial for DSB repair. 
     High throughput sequencing of the SELEX pools from various selection rounds (rounds 3, 5, 7, and 9—Panc-1 round 2), resulted in almost 8000 unique RNA families plus 78 ambiguous sequences, where RNA families are RNA sequences that differ by 4 nucleotides or less and ambiguous sequences are single RNA sequences that do not fit into a RNA family. The most representative sequence from each of the top 6 abundant families, designated Families A-F (FAM-A, etc., Tables 1-4), were transcribed to test their ability to bind to the nucleolin protein. Families B-F demonstrated specific binding to nucleolin while Family A did not appear to significantly bind the protein, suggesting that it may be an artifact resulting from PCR amplification ( FIG. 4 , Table 5). To make it easier to chemically synthesize the nucleolin aptamers, we sought to shorten their length. Thus, we designed truncates of the Families B-F aptamers (Tables 6-8). Several of these truncations resulted in improved affinity for nucleolin over the parent aptamers, with truncations Bvl, Dv2, Ev3, Ev5, and Fv3 demonstrating the best affinity ( FIGS. 5A-5F ). To further truncate the Ev3 aptamer, we designed 24 additional truncates of Ev3 (Tables 7 and 8). Several of these truncations, primarily Ev3.min21, Ev3.min22, and Ev3.min24 demonstrated a similar affinity for nucleolin compared to their parent Ev3 aptamer ( FIGS. 9A-9D ). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Nucleolin Aptamer Sequences without 5′ and 3′ 
               
               
                 Constant Regions 
               
            
           
           
               
               
            
               
                 NCL 
                   
               
               
                 Aptamer 
                 Sequence 
               
               
                   
               
               
                 FAM-A 
                 CCAUCUAGAUCUCCGUAGAUUCCCCCGGCUCUU 
               
               
                   
                 UCUCGC (SEQ ID NO: 1) 
               
               
                   
               
               
                 FAM-B 
                 AGCCAGCUUUGCAUACCACGUGCAAUUCACUCC 
               
               
                   
                 ACCCGUCA (SEQ ID NO: 2) 
               
               
                   
               
               
                 FAM-C 
                 AAGAUCUGCUAAGUGCACGCACAAUCACCAUCG 
               
               
                   
                 AGCGUCU (SEQ ID NO: 3) 
               
               
                   
               
               
                 FAM-D 
                 CACAUGGUACGCCCAAAGCGAGGCCCGCUGCGU 
               
               
                   
                 AGUGC (SEQ ID NO: 4) 
               
               
                   
               
               
                 FAM-E 
                 CACGGUCCAGCGCUAACUGUACCUGCUGUGCCA 
               
               
                   
                 CCCACCG (SEQ ID NO: 5) 
               
               
                   
               
               
                 FAM-F 
                 ACCACGCGCCAACGUGUCAGCUACACGCCGUGU 
               
               
                   
                 UCCCCGG (SEQ ID NO: 6) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Nucleolin Aptamer Sequences with 5′ and 3′ 
               
               
                 Constant Regions 
               
            
           
           
               
               
            
               
                 NCL 
                   
               
               
                 Aptamer 
                 Sequence 
               
               
                   
               
               
                 FAM-A 
                 GGGAGAGAGGAAGAGGGAUGGGCCAUCUAGAUCUC 
               
               
                   
                 CGUAGAUUCCCCCGGCUCUUUCUCGCCAUAACCCA 
               
               
                   
                 GAGGUCGAUAGUACUGGAUCCCCCC 
               
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
               
               
                 FAM-B 
                 GGGAGAGAGGAAGAGGGAUGGGAGCCAGCUUUGCA 
               
               
                   
                 UACCACGUGCAAUUCACUCCACCCGUCACAUAACC 
               
               
                   
                 CAGAGGUCGAUAGUACUGGAUCCCCCC 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
               
               
                 FAM-C 
                 GGGAGAGAGGAAGAGGGAUGGGAAGAUCUGCUAAG 
               
               
                   
                 UGCACGCACAAUCACCAUCGAGCGUCUCAUAACCC 
               
               
                   
                 AGAGGUCGAUAGUACUGGAUCCCCCC 
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
               
               
                 FAM-D 
                 GGGAGAGAGGAAGAGGGAUGGGCACAUGGUACGCC 
               
               
                   
                 CAAAGCGAGGCCCGCUGCGUAGUGCCAUAACCCAG 
               
               
                   
                 AGGUCGAUAGUACUGGAUCCCCCC 
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
               
               
                 FAM-E 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGCGC 
               
               
                   
                 UAACUGUACCUGCUGUGCCACCCACCGCAUAACCC 
               
               
                   
                 AGAGGUCGAUAGUACUGGAUCCCCCC 
               
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
               
               
                 FAM-F 
                 GGGAGAGAGGAAGAGGGAUGGGACCACGCGCCAAC 
               
               
                   
                 GUGUCAGCUACACGCCGUGUUCCCCGGCAUAACCC 
               
               
                   
                 AGAGGUCGAUAGUACUGGAUCCCCCC 
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Representative Nucleolin Aptamer Sequences 
               
               
                 without 5′ and 3′ Constant Regions from All 
               
               
                 Families 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                 Family 
                   
                 ID 
               
               
                 Name 
                 Representative Sequence 
                 NO: 
               
               
                   
               
               
                 A 
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 13 
               
               
                   
                 CGGCUCUUUCUCGC 
                   
               
               
                   
               
               
                 B 
                 AGCCAGCUUUGCAUACCACGUGCAA 
                 14 
               
               
                   
                 UUCACUCCACCCGUCA 
                   
               
               
                   
               
               
                 C 
                 AAGAUCUGCUAAGUGCACGCACAAU 
                 15 
               
               
                   
                 CACCAUCGAGCGUCU 
                   
               
               
                   
               
               
                 D 
                 CACAUGGUACGCCCAAAGCGAGGCC 
                 16 
               
               
                   
                 CGCUGCGUAGUGC 
                   
               
               
                   
               
               
                 E 
                 CACGGUCCAGCGCUAACUGUACCUG 
                 17 
               
               
                   
                 CUGUGCCACCCACCG 
                   
               
               
                   
               
               
                 F 
                 ACCACGCGCCAACGUGUCAGCUACA 
                 18 
               
               
                   
                 CGCCGUGUUCCCCGG 
                   
               
               
                   
               
               
                 G 
                 AAGAUCCUCGCGCAUCUGCCGAGCA 
                 19 
               
               
                   
                 AUCACCAUCGGACG 
                   
               
               
                   
               
               
                 H 
                 CCAAAUGCCAAGCCGUAGCCCGGCC 
                 20 
               
               
                   
                 AGUAGCCCACACGUC 
                   
               
               
                   
               
               
                 I 
                 UGCCAAGCCGAGGCCCGGCCACCAU 
                 21 
               
               
                   
                 CCACUGAUAGUGGGC 
                   
               
               
                   
               
               
                 J 
                 AAGAUCCUGACGCGACACAGCAAUC 
                 22 
               
               
                   
                 ACCAUCGAACCAGCU 
                   
               
               
                   
               
               
                 K 
                 AAGAUCUGCGGCAACGCACAAUCAC 
                 23 
               
               
                   
                 CAUCGAUUCCGAAUU 
                   
               
               
                   
               
               
                 L 
                 GAGCUCUCGAUUUCCUCCGCGACAC 
                 24 
               
               
                   
                 CCAUCCAAACCUCA 
                   
               
               
                   
               
               
                 M 
                 CUCUCCGGUCUACCAUCCGGACCGG 
                 25 
               
               
                   
                 CGACAAAGUCAACUU 
                   
               
               
                   
               
               
                 N 
                 AAGAUCUGCUAUGCACAAUCACCAU 
                 26 
               
               
                   
                 CGGGCGCUCCGGGGAA 
                   
               
               
                   
               
               
                 O 
                 UUGACUCUGCUGCGUAGUUCGCACC 
                 27 
               
               
                   
                 AAGAUCAACCACUUC 
                   
               
               
                   
               
               
                 P 
                 UACCAAGUCGUGGCCCGACUACCCA 
                 28 
               
               
                   
                 GCACGAUGCGCAA 
                   
               
               
                   
               
               
                 Q 
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 29 
               
               
                   
                 CAUCGAGAACCUAC 
                   
               
               
                   
               
               
                 R 
                 UGCCAAGCCGAGGCCCGGCCACCGU 
                 30 
               
               
                   
                 CCCCGCGGCUGAUGA 
                   
               
               
                   
               
               
                 S 
                 AAUGAUCUCGCCAAUGGGCGACAAU 
                 31 
               
               
                   
                 CACCAUGUCUUCACA 
                   
               
               
                   
               
               
                 T 
                 UCAGUGCGCCAAGUGGAGGCCCCAC 
                 32 
               
               
                   
                 CGCAGCCCAUCAA 
                   
               
               
                   
               
               
                 U 
                 UGUAUGCCAGCUUUGACGAUAACUG 
                 33 
               
               
                   
                 UCGCGCGUCAAUUCA 
                   
               
               
                   
               
               
                 V 
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 34 
               
               
                   
                 CCCCAUCAUGAGCUG 
                   
               
               
                   
               
               
                 W 
                 CCGCCAGCUUUGGGUACCCUGACCA 
                 35 
               
               
                   
                 AUUCACGGCCAUCCA 
                   
               
               
                   
               
               
                 X 
                 GUAAUUGUCUGAGACCACCGGACAA 
                 36 
               
               
                   
                 UCAACAAGAAAUCCU 
                   
               
               
                   
               
               
                 Y 
                 UCAGGCCAAAGUGUGAUAGCCACAC 
                 37 
               
               
                   
                 CCGCACCCAUCAGGA 
                   
               
               
                   
               
               
                 Z 
                 CCGACCGCCGACCAGGGUGCCACUC 
                 38 
               
               
                   
                 GUACCCCUGUCCGCC 
                   
               
               
                   
               
               
                 AA 
                 UGCCAAGUCGAAGCCCGACCACGCC 
                 39 
               
               
                   
                 AUCCCUAACAGUGCC 
                   
               
               
                   
               
               
                 AB 
                 ACUUGUGCUGAGUCGCCAAAGUGAG 
                 40 
               
               
                   
                 GCCCACUCGCCAGCA 
                   
               
               
                   
               
               
                 AC 
                 CCGCCAGCUCCUCUGAGGCACAAGA 
                 41 
               
               
                   
                 GGUUCACGGUGAUCC 
                   
               
               
                   
               
               
                 AD 
                 CACCAGGUUCUGCUGUCCCCAAGCG 
                 42 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
               
               
                 AE 
                 AAGAUCCGGUAACUCCCCACCGCAA 
                 43 
               
               
                   
                 UCACCGUCGACUACU 
                   
               
               
                   
               
               
                 AF 
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 44 
               
               
                   
                 CCGGCUCUUUCUCGC 
                   
               
               
                   
               
               
                 AG 
                 CCAUCUGAACCCACAGAUUCCCCCA 
                 45 
               
               
                   
                 UCAUCAGCCACAGUG 
                   
               
               
                   
               
               
                 AH 
                 CACUAAGUUGGUAGCCCCAACUGCC 
                 46 
               
               
                   
                 CCGACACGAGGAUGU 
                   
               
               
                   
               
               
                 AI 
                 UUGUGCUCCGUGGCUCCCCGGACCA 
                 47 
               
               
                   
                 ACCGCUUCCAGCAGU 
                   
               
               
                   
               
               
                 AJ 
                 CAAUCACGCGUAGUACGUCGCGGAA 
                 48 
               
               
                   
                 GAUCCCCAUGCCGA 
                   
               
               
                   
               
               
                 AK 
                 CACAUGGUACGCCCAAAAGCGAGGC 
                 49 
               
               
                   
                 CCGCUGCGUAGUGC 
                   
               
               
                   
               
               
                 AL 
                 UGCCAUACGCGGUUCGAAGUCGAAG 
                 50 
               
               
                   
                 CCCGACAACCCGGCA 
                   
               
               
                   
               
               
                 AM 
                 GUUAUUCACAUGCCUCCCGUGAAUC 
                 51 
               
               
                   
                 AACAAGAAUUCCUUG 
                   
               
               
                   
               
               
                 AN 
                 AAAGAUCUAGACUGUAAGUCUCCAA 
                 52 
               
               
                   
                 UCGCCCAGUUAAUUC 
                   
               
               
                   
               
               
                 AO 
                 GCCCAAUCGCCAGUGGAACGCGCUG 
                 53 
               
               
                   
                 AAGGAUCUGCACCC 
                   
               
               
                   
               
               
                 AP 
                 UGCAACGUAAAAGAGAGUCAUCUCA 
                 54 
               
               
                   
                 GGCUAGUCGUCUACC 
                   
               
               
                   
               
               
                 AQ 
                 GUGUACGCCAAGUCGAGGCCCGACC 
                 55 
               
               
                   
                 GUACCCAUACGCGAC 
                   
               
               
                   
               
               
                 AR 
                 UUAGCUCUACUUUCCUCUUCAGUAA 
                 56 
               
               
                   
                 GACUAACCGCUUCUU 
                   
               
               
                   
               
               
                 AS 
                 UCCAAGCGGAGGCCCCGCACCCACC 
                 57 
               
               
                   
                 CUCCAACGGGCACGG 
                   
               
               
                   
               
               
                 AT 
                 UAUCGCUCCACAACGACUCCCGUGG 
                 58 
               
               
                   
                 ACUACCCAAUUCCAA 
                   
               
               
                   
               
               
                 AU 
                 GUCGUGCCCAAGUGAAGGCCUCACG 
                 59 
               
               
                   
                 CACGCAUCCUAACCU 
                   
               
               
                   
               
               
                 AV 
                 AAGAUCUGCGCCAGCACAAUCACCA 
                 60 
               
               
                   
                 UCGUCCUGAGAAUGG 
                   
               
               
                   
               
               
                 AW 
                 AUGCCAAGCAGUGGCCCUGCCACCC 
                 61 
               
               
                   
                 ACCUAUCACUGUCGA 
                   
               
               
                   
               
               
                 AX 
                 AACAGACCAAGCAGCGGCCCUGCUC 
                 62 
               
               
                   
                 UGCCAUCAUACGCCU 
                   
               
               
                   
               
               
                 AY 
                 GUCAUUCGCUGACGAAUCAACAUGA 
                 63 
               
               
                   
                 AUUCCUAACUGCUGA 
                   
               
               
                   
               
               
                 AZ 
                 ACACGCCAAGCUGGUAGCCCCAGCC 
                 64 
               
               
                   
                 GUGCCCAUUACGGCC 
                   
               
               
                   
               
               
                 BA 
                 UAGCCAAGCAGCAGCCCUGCCAACC 
                 65 
               
               
                   
                 CAUCCUACCCGGGCG 
                   
               
               
                   
               
               
                 BB 
                 GCCCAAGGCGAGGCCCGCCGCUCCA 
                 66 
               
               
                   
                 UCCAGACGCUGAGGG 
                   
               
               
                   
               
               
                 BC 
                 AAGAUCUCGUCAUGCUUUGACGUCA 
                 67 
               
               
                   
                 AUCACCAUUGUUCCC 
                   
               
               
                   
               
               
                 BD 
                 AUCCCCCAGGAUGAGCACGUUGCCA 
                 68 
               
               
                   
                 UGGACUGGCUAUCC 
                   
               
               
                   
               
               
                 BE 
                 CUGUUACAGUCUCGCGUAACCCCCC 
                 69 
               
               
                   
                 CAUCGAUGUCCUCGA 
                   
               
               
                   
               
               
                 BF 
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 70 
               
               
                   
                 ACUCCACCCUGCUCA 
                   
               
               
                   
               
               
                 BG 
                 CACGGUAUAACCUCCUCAUAUACCU 
                 71 
               
               
                   
                 GCUGUGCCACCCGCG 
                   
               
               
                   
               
               
                 BH 
                 CCGGAAGAUCUGCUCGCACUAGCCG 
                 72 
               
               
                   
                 GAGCCCAAUCACGGC 
                   
               
               
                   
               
               
                 BI 
                 CCUGCCGAACGGCUAAGUCGCAGCC 
                 73 
               
               
                   
                 CGACCCGCGGCAGGG 
                   
               
               
                   
               
               
                 BJ 
                 CUCCGACCCGCGGACGAAGUCAACU 
                 74 
               
               
                   
                 UCCACAGUCCCACAC 
                   
               
               
                   
               
               
                 BK 
                 ACAUUAGGAUCUGCGUGAUGGGGAU 
                 75 
               
               
                   
                 CACCCGCUACAUGUC 
                   
               
               
                   
               
               
                 BL 
                 UCUAAGAUGGGGAAGAUCUCCGGAG 
                 76 
               
               
                   
                 CACCGGGCAAUCACC 
                   
               
               
                   
               
               
                 BM 
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 77 
               
               
                   
                 AUCGAGAACCUAC 
                   
               
               
                   
               
               
                 BN 
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 78 
               
               
                   
                 CCCUCACGAGAGAGG 
                   
               
               
                   
               
               
                 BO 
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 79 
               
               
                   
                 CCCGCCUGUGUGCUG 
                   
               
               
                   
               
               
                 BP 
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 80 
               
               
                   
                 AUCCAGAGUGUGAGA 
                   
               
               
                   
               
               
                 BQ 
                 AGCCAGCUUUUGCAUACCACGUGCA 
                 81 
               
               
                   
                 AUUCACUCCACCCGUCA 
                   
               
               
                   
               
               
                 BR 
                 CUUUGUAAACCCGGCAAACAAAAUC 
                 82 
               
               
                   
                 AACUUCCAUCAUCAA 
                   
               
               
                   
               
               
                 BS 
                 CCAUUGUAGCGACCACACAAUUCCC 
                 83 
               
               
                   
                 CAUCGGACAGCAUGG 
                   
               
               
                   
               
               
                 BT 
                 CUCUCGCCGUUCCCAGGCACGACAA 
                 84 
               
               
                   
                 AAUCAACUUCCCGCU 
                   
               
               
                   
               
               
                 BU 
                 AAGCCAAGCCGCGGCCCGGCCUUCC 
                 85 
               
               
                   
                 CAUGUGCUACUAGAG 
                   
               
               
                   
               
               
                 BV 
                 CCAAAUGCCAAAGCCGUAGCCCGGC 
                 86 
               
               
                   
                 CAGUAGCCCACACGUC 
                   
               
               
                   
               
               
                 BW 
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 87 
               
               
                   
                 CGUUUCCUCGUUAAG 
                   
               
               
                   
               
               
                 BX 
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 88 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
               
               
                 BY 
                 ACUGUCUGCAUACACGGUAUGCCCA 
                 89 
               
               
                   
                 ACGCCAUCCAAACCG 
                   
               
               
                   
               
               
                 BZ 
                 ACCUGCGGCUAUUGCCAGCGCCAUA 
                 90 
               
               
                   
                 AGACCCUCCACAGUA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Variant Nucleolin Aptamer Sequences without 
               
               
                 5′ and 3′ Constant Regions from All Families 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                 Family 
                   
                 ID 
               
               
                 Name 
                 All Family Sequences 
                 NO: 
               
               
                   
               
               
                 A 
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                  91 
               
               
                   
                 CGGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                  92 
               
               
                   
                 CGGCUCUUCCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                  93 
               
               
                   
                 CAGCUCUUUCUCGC 
                   
               
               
                   
               
               
                 B 
                 AGCCAGCUUUGCAUACCACGUGCAA 
                  94 
               
               
                   
                 UUCACUCCACCCGUCA 
                   
               
               
                   
                 AGCCAGCUUUGCAUACCACGUGCAA 
                  95 
               
               
                   
                 UUCACUCCACCCGUCG 
                   
               
               
                   
               
               
                 C 
                 AAGAUCUGCUAAGUGCACGCACAAU 
                  96 
               
               
                   
                 CACCAUCGAGCGUCU 
                   
               
               
                   
                 AAGAUCUGCUAAGUGCACGCACAAU 
                  97 
               
               
                   
                 CACCAUCGAGCGUCC 
                   
               
               
                   
                 AAGAUCUGCUAAGUGCACGCACAAU 
                  98 
               
               
                   
                 CACCAUCGAGCGCCU 
                   
               
               
                   
                 AAGAUCUGCUAAGUGCACGCACAAU 
                  99 
               
               
                   
                 CACCAUCGAGCGUC 
                   
               
               
                   
                 AAGAUCUGCUAAGUGCACGCACAAU 
                 100 
               
               
                   
                 CACCAUCGAGCGACU 
                   
               
               
                   
               
               
                 D 
                 CACAUGGUACGCCCAAAGCGAGGCC 
                 101 
               
               
                   
                 CGCUGCGUAGUGC 
                   
               
               
                   
                 CACACGGUACGCCCAAAGCGAGGCC 
                 102 
               
               
                   
                 CGCUGCGUAGUGC 
                   
               
               
                   
               
               
                 E 
                 CACGGUCCAGCGCUAACUGUACCUG 
                 103 
               
               
                   
                 CUGUGCCACCCACCG 
                   
               
               
                   
                 CACGGUCCAGCGCUAACUGUACCUG 
                 104 
               
               
                   
                 CUGUGCCACCCACCA 
                   
               
               
                   
                 CACGGUCCAGCGCUAACUGUACCUG 
                 105 
               
               
                   
                 CUGUGCCACCCACUG 
                   
               
               
                   
                 CACGGUCCAGCGCUAACUGUACCUG 
                 106 
               
               
                   
                 CUGUGCCACCCACCU 
                   
               
               
                   
                 CACGGUCCAGCGCUAACUGUACCUG 
                 107 
               
               
                   
                 CUGUGCCACCCGCCG 
                   
               
               
                   
               
               
                 F 
                 ACCACGCGCCAACGUGUCAGCUACA 
                 108 
               
               
                   
                 CGCCGUGUUCCCCGG 
                   
               
               
                   
                 ACCACGCGCCAACGUGUCAGCUACA 
                 109 
               
               
                   
                 CGCCGUGUUCCCCGA 
                   
               
               
                   
                 ACCACGCGCCAACGUGUCAGCUACA 
                 110 
               
               
                   
                 CGCCGUGUUCCCCG 
                   
               
               
                   
                 CCACGCGCCAACGUGUCAGCUACAC 
                 111 
               
               
                   
                 GCCGUGUUCCCCGG 
                   
               
               
                   
               
               
                 G 
                 AAGAUCCUCGCGCAUCUGCCGAGCA 
                 112 
               
               
                   
                 AUCACCAUCGGACG 
                   
               
               
                   
                 AAGAUCCUCGCGCAUCUGCCGAGCA 
                 113 
               
               
                   
                 AUCACCAUCGGACC 
                   
               
               
                   
                 AAGAUCCUCGCGCAUCUGCCGAGCA 
                 114 
               
               
                   
                 AUCACCAUCGGACA 
                   
               
               
                   
                 AAGAUCCUCGCGCAUCUGCCGAGCA 
                 115 
               
               
                   
                 AUCACCAUCGGACU 
                   
               
               
                   
                 AAAGAUCCUCGCGCAUCUGCCGAGC 
                 116 
               
               
                   
                 AAUCACCAUCGGACG 
                   
               
               
                   
                 AAGAUCCUCGCGCACCUGCCGAGCA 
                 117 
               
               
                   
                 AUCACCAUCGGACG 
                   
               
               
                   
               
               
                 H 
                 CCAAAUGCCAAGCCGUAGCCCGGCC 
                 118 
               
               
                   
                 AGUAGCCCACACGUC 
                   
               
               
                   
                 CCAAAAUGCCAAGCCGUAGCCCGGC 
                 119 
               
               
                   
                 CAGUAGCCCACACGUC 
                   
               
               
                   
                 CCAAAUGCCAAGCCGUAGCCCGGCC 
                 120 
               
               
                   
                 AGUAGCCCACACGAC 
                   
               
               
                   
                 CCAAAUGCCAAGCCGUAGCCCGGCC 
                 121 
               
               
                   
                 AGUAGCCCACACGUA 
                   
               
               
                   
               
               
                 I 
                 UGCCAAGCCGAGGCCCGGCCACCAU 
                 122 
               
               
                   
                 CCACUGAUAGUGGGC 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCACCAU 
                 123 
               
               
                   
                 CCACUGAUAGUGGGA 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCACCAU 
                 124 
               
               
                   
                 CCACUGAUAGUGGG 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCACCAU 
                 125 
               
               
                   
                 CCACUGAUAGUGGGU 
                   
               
               
                   
               
               
                 J 
                 AAGAUCCUGACGCGACACAGCAAUC 
                 126 
               
               
                   
                 ACCAUCGAACCAGCU 
                   
               
               
                   
                 AAGAUCCUGACGCGACACAGCAAUC 
                 127 
               
               
                   
                 ACCAUCGAACCAGCC 
                   
               
               
                   
               
               
                 K 
                 AAGAUCUGCGGCAACGCACAAUCAC 
                 128 
               
               
                   
                 CAUCGAUUCCGAAUU 
                   
               
               
                   
                 AAGAUCUGCGGCAACGCACAAUCAC 
                 129 
               
               
                   
                 CAUCGAUUCCGAAUG 
                   
               
               
                   
                 AAGAUCUGCGGCAACGCACAAUCAC 
                 130 
               
               
                   
                 CAUCGAUUCCGAAUC 
                   
               
               
                   
                 AAGAUCUGCGGCAACGCACAAUCAC 
                 131 
               
               
                   
                 CAUCGAUUCCGAACU 
                   
               
               
                   
                 AAGAUCUGCGGCAACGUACAAUCAC 
                 132 
               
               
                   
                 CAUCGAUUCCGAAUU 
                   
               
               
                   
               
               
                 L 
                 GAGCUCUCGAUUUCCUCCGCGACAC 
                 133 
               
               
                   
                 CCAUCCAAACCUCA 
                   
               
               
                   
                 AGCUCUCGAUUUCCUCCGCGACACC 
                 134 
               
               
                   
                 CAUCCAAACCUCA 
                   
               
               
                   
                 GAGCUCUCGAUUUCCUCCGCGACAC 
                 135 
               
               
                   
                 CCAUCCAAACCUCG 
                   
               
               
                   
               
               
                 M 
                 CUCUCCGGUCUACCAUCCGGACCGG 
                 136 
               
               
                   
                 CGACAAAGUCAACUU 
                   
               
               
                   
                 CUCUCCGGUCUACCACCCGGACCGG 
                 137 
               
               
                   
                 CGACAAAGUCAACUU 
                   
               
               
                   
               
               
                 N 
                 AAGAUCUGCUAUGCACAAUCACCAU 
                 138 
               
               
                   
                 CGGGCGCUCCGGGGAA 
                   
               
               
                   
                 AAGAUCUGCUAUGCACAAUCACCAU 
                 139 
               
               
                   
                 CGGGCGCUCCGGGAA 
                   
               
               
                   
                 AAGAUCUGCUACGCACAAUCACCAU 
                 140 
               
               
                   
                 CGGGCGCUCCGGGGAA 
                   
               
               
                   
               
               
                 O 
                 UUGACUCUGCUGCGUAGUUCGCACC 
                 141 
               
               
                   
                 AAGAUCAACCACUUC 
                   
               
               
                   
                 UUGACUCUGCUGCGUAGUUCGCACC 
                 142 
               
               
                   
                 AAGAUCAACCACUUCC 
                   
               
               
                   
                 UUGACUCUGCUGCGUAGCUCGCACC 
                 143 
               
               
                   
                 AAGAUCAACCACUUC 
                   
               
               
                   
                 UUGACUCUGCUGCGCAGUUCGCACC 
                 144 
               
               
                   
                 AAGAUCAACCACUUC 
                   
               
               
                   
                 UUGACUCUGCUGCGUAGUCCGCACC 
                 145 
               
               
                   
                 AAGAUCAACCACUUC 
                   
               
               
                   
               
               
                 P 
                 UACCAAGUCGUGGCCCGACUACCCA 
                 146 
               
               
                   
                 GCACGAUGCGCAA 
                   
               
               
                   
                 UACCAAAGUCGUGGCCCGACUACCC 
                 147 
               
               
                   
                 AGCACGAUGCGCAA 
                   
               
               
                   
                 UACCAAGUCGUGGCCCGACUACCCA 
                 148 
               
               
                   
                 GCACGGUGCGCAA 
                   
               
               
                   
                 UACCAAGUCGUGGCCCGACUACCCA 
                 149 
               
               
                   
                 GCACGAUGCGCAG 
                   
               
               
                   
                 UACCAAGUCGUGGCCCGACUACCCA 
                 150 
               
               
                   
                 GCACAAUGCGCAA 
                   
               
               
                   
                 UACCAAGUCGCGGCCCGACUACCCA 
                 151 
               
               
                   
                 GCACGAUGCGCAA 
                   
               
               
                   
               
               
                 Q 
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 152 
               
               
                   
                 CAUCGAGAACCUAC 
                   
               
               
                   
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 153 
               
               
                   
                 CAUCGAGAACCUA 
                   
               
               
                   
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 154 
               
               
                   
                 CAUCGAGAACCUAU 
                   
               
               
                   
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 155 
               
               
                   
                 CAUCGAGAACCUAA 
                   
               
               
                   
               
               
                 R 
                 UGCCAAGCCGAGGCCCGGCCACCGU 
                 156 
               
               
                   
                 CCCCGCGGCUGAUGA 
                   
               
               
                   
                 UGCCAAAGCCGAGGCCCGGCCACCG 
                 157 
               
               
                   
                 UCCCCGCGGCUGAUGA 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCACCGU 
                 158 
               
               
                   
                 CCCCGCGGCUGAUCGA 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCACCGU 
                 159 
               
               
                   
                 CCCCGCGGCUGAUGG 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCACCGU 
                 160 
               
               
                   
                 CCCCGCGGCUGACGA 
                   
               
               
                   
               
               
                 S 
                 AAUGAUCUCGCCAAUGGGCGACAAU 
                 161 
               
               
                   
                 CACCAUGUCUUCACA 
                   
               
               
                   
                 AACGAUCUCGCCAAUGGGCGACAAU 
                 162 
               
               
                   
                 CACCAUGUCUUCACA 
                   
               
               
                   
                 AAUGAUCUCGCCAAUGGGCGACAAU 
                 163 
               
               
                   
                 CACCAUGUCUUCACG 
                   
               
               
                   
                 AAUGAUCUCGCCAAUGUGCGACAAU 
                 164 
               
               
                   
                 CACCAUGUCUUCACA 
                   
               
               
                   
               
               
                 T 
                 UCAGUGCGCCAAGUGGAGGCCCCAC 
                 165 
               
               
                   
                 CGCAGCCCAUCAA 
                   
               
               
                   
                 UCAGUGCGCCAAGUGGAGGCCCCAC 
                 166 
               
               
                   
                 CGCAGCCCAUCGA 
                   
               
               
                   
                 UCAGUGCGCCAAGUGGAGGCCCCAC 
                 167 
               
               
                   
                 CGCAGCCCAUCAG 
                   
               
               
                   
               
               
                 U 
                 UGUAUGCCAGCUUUGACGAUAACUG 
                 168 
               
               
                   
                 UCGCGCGUCAAUUCA 
                   
               
               
                   
               
               
                 V 
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 169 
               
               
                   
                 CCCCAUCAUGAGCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 170 
               
               
                   
                 CCCCAUCAUGAGCCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 171 
               
               
                   
                 CCCCAUCAUGAGCUC 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 172 
               
               
                   
                 CCCCAUCAUGGGCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 173 
               
               
                   
                 UCCCAUCAUGAGCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 174 
               
               
                   
                 CCCCAUCGUGAGCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 175 
               
               
                   
                 CUCCAUCAUGAGCUG 
                   
               
               
                   
                 CACGCCAAAGUGGAGCCCACUCGUA 
                 176 
               
               
                   
                 CCCCAUCAUGAGCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGCA 
                 177 
               
               
                   
                 CCCCAUCAUGAGCUG 
                   
               
               
                   
                 UACGCCAAAGUGGAGCCCACUCGUA 
                 178 
               
               
                   
                 CCCCAUCAUGAGCUA 
                   
               
               
                   
               
               
                 W 
                 CCGCCAGCUUUGGGUACCCUGACCA 
                 179 
               
               
                   
                 AUUCACGGCCAUCCA 
                   
               
               
                   
                 CCGCCAGCUUUGGGUACCCUGACCA 
                 180 
               
               
                   
                 AUUCACGGCCAUCCG 
                   
               
               
                   
                 CCGCCCAGCUUUGGGUACCCUGACC 
                 181 
               
               
                   
                 AAUUCACGGCCAUCCA 
                   
               
               
                   
               
               
                 X 
                 GUAAUUGUCUGAGACCACCGGACAA 
                 182 
               
               
                   
                 UCAACAAGAAAUCCU 
                   
               
               
                   
                 GUAAUUGUCUGAGACCACCGGACAA 
                 183 
               
               
                   
                 UCAACAAGAAAAUCCU 
                   
               
               
                   
                 UAAUUGUCUGAGACCACCGGACAAU 
                 184 
               
               
                   
                 CAACAAGAAAUCCU 
                   
               
               
                   
               
               
                 Y 
                 UCAGGCCAAAGUGUGAUAGCCACAC 
                 185 
               
               
                   
                 CCGCACCCAUCAGGA 
                   
               
               
                   
                 UCAGGCCAAAGUGUGAUAGCCACAC 
                 186 
               
               
                   
                 CCGCACCCAUCAGA 
                   
               
               
                   
                 UCAGGCCAAAGUGUGAUAGCCACAC 
                 187 
               
               
                   
                 CCGCACCCAUCAGG 
                   
               
               
                   
               
               
                 Z 
                 CCGACCGCCGACCAGGGUGCCACUC 
                 188 
               
               
                   
                 GUACCCCUGUCCGCC 
                   
               
               
                   
                 CCGACCGCCGACCAGGGUGCCACUC 
                 189 
               
               
                   
                 GUACCCCUGUCCGCCC 
                   
               
               
                   
                 CCGACCGCCGACCAGGGUGCCACUC 
                 190 
               
               
                   
                 GUACCCCUGUCCCGCC 
                   
               
               
                   
                 CCGACCGCCGACCAGGGUGCCACUC 
                 191 
               
               
                   
                 GUACCCCUGUCCGC 
                   
               
               
                   
               
               
                 AA 
                 UGCCAAGUCGAAGCCCGACCACGCC 
                 192 
               
               
                   
                 AUCCCUAACAGUGCC 
                   
               
               
                   
                 UGCCAAAGUCGAAGCCCGACCACGC 
                 193 
               
               
                   
                 CAUCCCUAACAGUGCC 
                   
               
               
                   
                 UGCCAAGUCGAAGCCCGACCACGCC 
                 194 
               
               
                   
                 AUCCCUAACAGUGC 
                   
               
               
                   
                 UGCCAAGUCGAAGCCCGACCACGCC 
                 195 
               
               
                   
                 AUCCCUAACGGUGCC 
                   
               
               
                   
                 UGCCAAGUCGAAGCCCGACCACGCC 
                 196 
               
               
                   
                 AUCCCUAACAGUGCA 
                   
               
               
                   
                 UGCCAAGUCGAGGCCCGACCACGCC 
                 197 
               
               
                   
                 AUCCCUAACAGUGCC 
                   
               
               
                   
                 UGCCAAGCCGAAGCCCGACCACGCC 
                 198 
               
               
                   
                 AUCCCUAACAGUGCC 
                   
               
               
                   
               
               
                 AB 
                 ACUUGUGCUGAGUCGCCAAAGUGAG 
                 199 
               
               
                   
                 GCCCACUCGCCAGCA 
                   
               
               
                   
                 GCUUGUGCUGAGUCGCCAAAGUGAG 
                 200 
               
               
                   
                 GCCCACUCGCCAGCA 
                   
               
               
                   
                 ACCUGUGCUGAGUCGCCAAAGUGAG 
                 201 
               
               
                   
                 GCCCACUCGCCAGCA 
                   
               
               
                   
               
               
                 AC 
                 CCGCCAGCUCCUCUGAGGCACAAGA 
                 202 
               
               
                   
                 GGUUCACGGUGAUCC 
                   
               
               
                   
                 CCGCCAGCUCCUCUGAGGCACAAGA 
                 203 
               
               
                   
                 GGUUCACGGUGAUCCC 
                   
               
               
                   
               
               
                 AD 
                 CACCAGGUUCUGCUGUCCCCAAGCG 
                 204 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
                 CACCAGGUUCUGCUAUCCCCAAGCG 
                 205 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
                 CACCAGGUUCUGCUGUCUCCAAGCG 
                 206 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
                 CACCAGGUUCUGCUGUUCCCAAGCG 
                 207 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
                 CACCAGGUCCUGCUGUCCCCAAGCG 
                 208 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
                 CACCAGGCUCUGCUGUCCCCAAGCG 
                 209 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
                 CACCAGGUUCUGCUGUCCUCAAGCG 
                 210 
               
               
                   
                 CUGACCCAUCCUUCC 
                   
               
               
                   
               
               
                 AE 
                 AAGAUCCGGUAACUCCCCACCGCAA 
                 211 
               
               
                   
                 UCACCGUCGACUACU 
                   
               
               
                   
                 AAGAUCCGGUGACUCCCCACCGCAA 
                 212 
               
               
                   
                 UCACCGUCGACUACU 
                   
               
               
                   
                 AAGAUCCGGUAACUCCCUACCGCAA 
                 213 
               
               
                   
                 UCACCGUCGACUACU 
                   
               
               
                   
                 AAAGAUCCGGUAACUCCCCACCGCA 
                 214 
               
               
                   
                 AUCACCGUCGACUACU 
                   
               
               
                   
               
               
                 AF 
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 215 
               
               
                   
                 CCGGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 216 
               
               
                   
                 CGGGCUCUUUCUCGU 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 217 
               
               
                   
                 CGGGCUCUUUCUCGA 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 218 
               
               
                   
                 CGGGCUCUUUCUCG 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 219 
               
               
                   
                 CGGGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 220 
               
               
                   
                 CGGGCUCUUUCUCAC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUUCCC 
                 221 
               
               
                   
                 CCGGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 222 
               
               
                   
                 CGGGCUCUUCCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 223 
               
               
                   
                 CGGGCUCUCUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 224 
               
               
                   
                 CGGGCUCUUUCUUGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 225 
               
               
                   
                 CGGGCCCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 226 
               
               
                   
                 CGGGCUCUUUCUCUC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 227 
               
               
                   
                 CGGCCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 228 
               
               
                   
                 CGGGCUCUUUCUCCC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 229 
               
               
                   
                 CGGGCUCUUUCUCGUC 
                   
               
               
                   
               
               
                 AG 
                 CCAUCUGAACCCACAGAUUCCCCCA 
                 230 
               
               
                   
                 UCAUCAGCCACAGUG 
                   
               
               
                   
                 CCAUCUGAACCCACAGAUUCCCCCA 
                 231 
               
               
                   
                 UCAUCAGCCACAGUA 
                   
               
               
                   
                 CCAUCUGAACCCACAGAUUCCCCCA 
                 232 
               
               
                   
                 UCAUCAGCCACAGCG 
                   
               
               
                   
                 CCAUCUGAACCCACAGAUUCCCCCA 
                 233 
               
               
                   
                 UCAUCAGCCACAGUC 
                   
               
               
                   
                 CCAUCUGAACCCACAGAUUCCCCCA 
                 234 
               
               
                   
                 UCAUCAGCCACGGUG 
                   
               
               
                   
               
               
                 AH 
                 CACUAAGUUGGUAGCCCCAACUGCC 
                 235 
               
               
                   
                 CCGACACGAGGAUGU 
                   
               
               
                   
                 CACUAAGUUGGUAGCCCCAACUGCC 
                 236 
               
               
                   
                 CCGACACGAGGAUGUC 
                   
               
               
                   
                 CACUAAGUUGGUAGCCCCAACUGCC 
                 237 
               
               
                   
                 CCGACACGAGGAUGC 
                   
               
               
                   
               
               
                 AI 
                 UUGUGCUCCGUGGCUCCCCGGACCA 
                 238 
               
               
                   
                 ACCGCUUCCAGCAGU 
                   
               
               
                   
                 UUGUGUUCCGUGGCUCCCCGGACCA 
                 239 
               
               
                   
                 ACCGCUUCCAGCAGU 
                   
               
               
                   
                 UUGUGCUCCGUGGCUCCCCGGACCA 
                 240 
               
               
                   
                 ACCGCUUCCAGCAGC 
                   
               
               
                   
                 UUGCGCUCCGUGGCUCCCCGGACCA 
                 241 
               
               
                   
                 ACCGCUUCCAGCAGU 
                   
               
               
                   
               
               
                 AJ 
                 CAAUCACGCGUAGUACGUCGCGGAA 
                 242 
               
               
                   
                 GAUCCCCAUGCCGA 
                   
               
               
                   
                 CAAUCACGCGUAGUACGUCGCGGAA 
                 243 
               
               
                   
                 GAUCCCCAUGCCGG 
                   
               
               
                   
                 CAAUCACGCGUAGUACGUCGCGGAA 
                 244 
               
               
                   
                 GAUCCCCAUGCCAA 
                   
               
               
                   
                 CAAUCACGCGUAGUACGUCGCGGAA 
                 245 
               
               
                   
                 GAUCCCCAUGCCGU 
                   
               
               
                   
                 CAAUCACGCGUAGCACGUCGCGGAA 
                 246 
               
               
                   
                 GAUCCCCAUGCCGA 
                   
               
               
                   
                 CAAUCACGCGUAGUACGUCGCGGAG 
                 247 
               
               
                   
                 GAUCCCCAUGCCGA 
                   
               
               
                   
               
               
                 AK 
                 CACAUGGUACGCCCAAAAGCGAGGC 
                 248 
               
               
                   
                 CCGCUGCGUAGUGC 
                   
               
               
                   
                 CACAUGGUACGCCCCAAAGCGAGGC 
                 249 
               
               
                   
                 CCGCUGCGUAGUGC 
                   
               
               
                   
                 CACAUGGUACGCCCAAAGCCGAGGC 
                 250 
               
               
                   
                 CCGCUGCGUAGUGC 
                   
               
               
                   
                 CACAUGGUACGCCCAAAAGCGAGGC 
                 251 
               
               
                   
                 CCGCUGCGUAGUG 
                   
               
               
                   
               
               
                 AL 
                 UGCCAUACGCGGUUCGAAGUCGAAG 
                 252 
               
               
                   
                 CCCGACAACCCGGCA 
                   
               
               
                   
                 UGCCAUACGCGGUUCGAAGUCGAAG 
                 253 
               
               
                   
                 CCCGACAACCCCGGCA 
                   
               
               
                   
                 UGCCAUACGCGGUUCGAAGUCGAGG 
                 254 
               
               
                   
                 CCCGACAACCCGGCA 
                   
               
               
                   
               
               
                 AM 
                 GUUAUUCACAUGCCUCCCGUGAAUC 
                 255 
               
               
                   
                 AACAAGAAUUCCUUG 
                   
               
               
                   
                 UUAUUCACAUGCCUCCCGUGAAUCA 
                 256 
               
               
                   
                 ACAAGAAUUCCUUG 
                   
               
               
                   
                 GUUAUUCACAUGCCUCCCGUGAAUC 
                 257 
               
               
                   
                 AACAAGAAUUCCUCG 
                   
               
               
                   
                 GUUAUUCACAUGCCUCUCGUGAAUC 
                 258 
               
               
                   
                 AACAAGAAUUCCUUG 
                   
               
               
                   
                   
                 259 
               
               
                   
               
               
                 AN 
                 AAAGAUCUAGACUGUAAGUCUCCAA 
                 260 
               
               
                   
                 UCGCCCAGUUAAUUC 
                   
               
               
                   
                 AAAAGAUCUAGACUGUAAGUCUCCA 
                 261 
               
               
                   
                 AUCGCCCAGUUAAUUC 
                   
               
               
                   
                 AAAGAUCUAGACUGUAAGUCUCCAA 
                 262 
               
               
                   
                 UCGCCCAGUAAUUC 
                   
               
               
                   
               
               
                 AO 
                 GCCCAAUCGCCAGUGGAACGCGCUG 
                 263 
               
               
                   
                 AAGGAUCUGCACCC 
                   
               
               
                   
                 GCCCAAUCGCCAGUGGAACGCGCUG 
                 264 
               
               
                   
                 AAGGAUCUGCACC 
                   
               
               
                   
                 GCCCAAUCGCCAGUGGAACGCACUG 
                 265 
               
               
                   
                 AAGGAUCUGCACCC 
                   
               
               
                   
                 GCCCAAUCGCCAGUGGAACGCGCUG 
                 266 
               
               
                   
                 AAGGAUCUGCACCCC 
                   
               
               
                   
                 CCCAAUCGCCAGUGGAACGCGCUGA 
                 267 
               
               
                   
                 AGGAUCUGCACCC 
                   
               
               
                   
                 GCCCAAUCGCCAGCGGAACGCGCUG 
                 268 
               
               
                   
                 AAGGAUCUGCACCC 
                   
               
               
                   
               
               
                 AP 
                 UGCAACGUAAAAGAGAGUCAUCUCA 
                 269 
               
               
                   
                 GGCUAGUCGUCUACC 
                   
               
               
                   
                 UGCAACGUAAAAGAGAGUCAUCUCA 
                 270 
               
               
                   
                 GGCUAGUCGUCUAC 
                   
               
               
                   
               
               
                 AQ 
                 GUGUACGCCAAGUCGAGGCCCGACC 
                 271 
               
               
                   
                 GUACCCAUACGCGAC 
                   
               
               
                   
                 UGUACGCCAAGUCGAGGCCCGACCG 
                 272 
               
               
                   
                 UACCCAUACGCGAC 
                   
               
               
                   
                 GUGUACGCCAAGUCGAGGCCCGACC 
                 273 
               
               
                   
                 GUACCCAUACGCGGC 
                   
               
               
                   
                 GUGUACGCCAAGUCGAGGCCCGACC 
                 274 
               
               
                   
                 GUACCCAUACGCGAU 
                   
               
               
                   
               
               
                 AR 
                 UUAGCUCUACUUUCCUCUUCAGUAA 
                 275 
               
               
                   
                 GACUAACCGCUUCUU 
                   
               
               
                   
                 UUAGCUCUACUUUCCUCUUCAGUAA 
                 276 
               
               
                   
                 GACUAACCGCUUCCU 
                   
               
               
                   
                 UUAGCUCUACUUUCCUCUUCAGUAA 
                 277 
               
               
                   
                 GACUAACCGCUUCUC 
                   
               
               
                   
                 UUAGCUCUACUUUCCUCUUCAGUAA 
                 278 
               
               
                   
                 GACUAACCGCUCCUU 
                   
               
               
                   
               
               
                 AS 
                 UCCAAGCGGAGGCCCCGCACCCACC 
                 279 
               
               
                   
                 CUCCAACGGGCACGG 
                   
               
               
                   
                 UCCAAGCGGAGGCCCCGCACCCACC 
                 280 
               
               
                   
                 CUCCAACGGGCACGC 
                   
               
               
                   
                 UCCAAGCGGAGGCCCCGUACCCACC 
                 281 
               
               
                   
                 CUCCAACGGGCACGG 
                   
               
               
                   
                 UCCAAGCGGAGGCCCCGCACCCACC 
                 282 
               
               
                   
                 CCCCAACGGGCACGG 
                   
               
               
                   
                 UCCAAGCGGAGGCCCCGCACCCACC 
                 283 
               
               
                   
                 CUCCAACGGGCACGA 
                   
               
               
                   
                 UCCAAAGCGGAGGCCCCGCACCCAC 
                 284 
               
               
                   
                 CCUCCAACGGGCACGG 
                   
               
               
                   
                 UCCAAGCGGAGGCCCCGCACCCACC 
                 285 
               
               
                   
                 CUCCAACGGGCACAG 
                   
               
               
                   
               
               
                 AT 
                 UAUCGCUCCACAACGACUCCCGUGG 
                 286 
               
               
                   
                 ACUACCCAAUUCCAA 
                   
               
               
                   
                 UAUCGCUCCACAACGACUCCCGUGG 
                 287 
               
               
                   
                 ACUACCCAAUUCCAG 
                   
               
               
                   
                 UAUCGCUCCACAACGACUCCCGUGG 
                 288 
               
               
                   
                 ACUACCCAAUUCCAAA 
                   
               
               
                   
                 UAUCGCUCCACAACGACUCCCGUGG 
                 289 
               
               
                   
                 ACUACCCAAUUCCAU 
                   
               
               
                   
               
               
                 AU 
                 GUCGUGCCCAAGUGAAGGCCUCACG 
                 290 
               
               
                   
                 CACGCAUCCUAACCU 
                   
               
               
                   
                 UCGUGCCCAAGUGAAGGCCUCACGC 
                 291 
               
               
                   
                 ACGCAUCCUAACCU 
                   
               
               
                   
                 GUCGUGCCCAAGUGAAGGCCUCACG 
                 292 
               
               
                   
                 CACGCAUCCUAACCC 
                   
               
               
                   
               
               
                 AV 
                 AAGAUCUGCGCCAGCACAAUCACCA 
                 293 
               
               
                   
                 UCGUCCUGAGAAUGG 
                   
               
               
                   
                 AAGAUCUGCGCCAGCACAAUCACCA 
                 294 
               
               
                   
                 UCGUCCUGAGAAUGC 
                   
               
               
                   
                 AAGAUCUGCGCCAGCACAAUCACCA 
                 295 
               
               
                   
                 UCGUCCUGAGAAUGA 
                   
               
               
                   
                 AAGAUCUGCGCCAGCACAAUCACCA 
                 296 
               
               
                   
                 UCGUCCUGAGAGUGG 
                   
               
               
                   
                 AAGAUCUGCGCCAGCACAAUCACCA 
                 297 
               
               
                   
                 UCGUCCUGGGAAUGG 
                   
               
               
                   
               
               
                 AW 
                 AUGCCAAGCAGUGGCCCUGCCACCC 
                 298 
               
               
                   
                 ACCUAUCACUGUCGA 
                   
               
               
                   
                 AUGCCAAGCAGUCGGCCUGCCACCC 
                 299 
               
               
                   
                 ACCUAUCACUGUCGA 
                   
               
               
                   
                 AUGCCAAGCAGUGGCCCUGCCACCC 
                 300 
               
               
                   
                 ACCUAUCACUAUCGA 
                   
               
               
                   
                 AUGCCAAGCAGUGGCCCUGCCACCC 
                 301 
               
               
                   
                 ACCUACCACUGUCGA 
                   
               
               
                   
                 AUGCCAAGCAGCGGCCCUGCCACCC 
                 302 
               
               
                   
                 ACCUAUCACUGUCGA 
                   
               
               
                   
               
               
                 AX 
                 AACAGACCAAGCAGCGGCCCUGCUC 
                 303 
               
               
                   
                 UGCCAUCAUACGCCU 
                   
               
               
                   
                 GACAGACCAAGCAGCGGCCCUGCUC 
                 304 
               
               
                   
                 UGCCAUCAUACGCCU 
                   
               
               
                   
                 AACAGACCAAGCAGUGGCCCUGCUC 
                 305 
               
               
                   
                 UGCCAUCAUACGCCU 
                   
               
               
                   
                 AACAGACCAAGCAGCGGCCCUGCUC 
                 306 
               
               
                   
                 UGCCAUCAUACGCCC 
                   
               
               
                   
                 AACAGACCAAGCAGCGGCCCUGCUC 
                 307 
               
               
                   
                 UGCCAUCAUACACCU 
                   
               
               
                   
                 ACAGACCAAGCAGCGGCCCUGCUCU 
                 308 
               
               
                   
                 GCCAUCAUACGCCU 
                   
               
               
                   
                 AACAGACCAAGCAGCGGCCCUGCUC 
                 309 
               
               
                   
                 UGCCAUCAUACGCCCU 
                   
               
               
                   
               
               
                 AY 
                 GUCAUUCGCUGACGAAUCAACAUGA 
                 310 
               
               
                   
                 AUUCCUAACUGCUGA 
                   
               
               
                   
                 UCAUUCGCUGACGAAUCAACAUGAA 
                 311 
               
               
                   
                 UUCCUAACUGCUGA 
                   
               
               
                   
                 GUCAUUCGCUGACGAAUCAACAUGA 
                 312 
               
               
                   
                 AUUCCUAACUGCCGA 
                   
               
               
                   
                 GUCAUUCGCUGACGAAUCAACAUGA 
                 313 
               
               
                   
                 AUUCCUAACUGCUGG 
                   
               
               
                   
               
               
                 AZ 
                 ACACGCCAAGCUGGUAGCCCCAGCC 
                 314 
               
               
                   
                 GUGCCCAUUACGGCC 
                   
               
               
                   
                 ACACGCCAAGCUGGUAGCCCCAGCC 
                 315 
               
               
                   
                 GUGCCCAUUACGGC 
                   
               
               
                   
                 ACACGCCAAGCUGGUAGCCCCAGCC 
                 316 
               
               
                   
                 GUGCCCAUUACGGUC 
                   
               
               
                   
                 ACACGCCAAGCUGGUAGCCCCAGCC 
                 317 
               
               
                   
                 GUACCCAUUACGGCC 
                   
               
               
                   
               
               
                 BA 
                 UAGCCAAGCAGCAGCCCUGCCAACC 
                 318 
               
               
                   
                 CAUCCUACCCGGGCG 
                   
               
               
                   
                 UAGCCAAGCAGCAGCCCUGCCAACC 
                 319 
               
               
                   
                 CAUCCUACCCGGCG 
                   
               
               
                   
                 UAGCCAAGCAGCAGCCCUGCCAACC 
                 320 
               
               
                   
                 CAUCCUACCCGGGCA 
                   
               
               
                   
                 UAGCCAAGCAGCAGCCCUGCCAACC 
                 321 
               
               
                   
                 CAUCCUACCCGGGUG 
                   
               
               
                   
                 UAGCCAAGCAGCGGCCCUGCCAACC 
                 322 
               
               
                   
                 CAUCCUACCCGGGCG 
                   
               
               
                   
               
               
                 BB 
                 GCCCAAGGCGAGGCCCGCCGCUCCA 
                 323 
               
               
                   
                 UCCAGACGCUGAGGG 
                   
               
               
                   
                 GCCCAAGGCGAGGCCCGCCGCUCCA 
                 324 
               
               
                   
                 UCCAGACGCUGAGG 
                   
               
               
                   
                 CCCAAGGCGAGGCCCGCCGCUCCAU 
                 325 
               
               
                   
                 CCAGACGCUGAGGG 
                   
               
               
                   
                 CCCAAGGCGAGGCCCGCCGCUCCAU 
                 326 
               
               
                   
                 CCAGACGCUGAGG 
                   
               
               
                   
                 GCCCAAGGCGAGGCCCGCCGCUCCA 
                 327 
               
               
                   
                 UCCAGACGCUGAGGC 
                   
               
               
                   
                 GCCCAAAGGCGAGGCCCGCCGCUCC 
                 328 
               
               
                   
                 AUCCAGACGCUGAGGG 
                   
               
               
                   
                 GCCCAAGGCGAGGCCCGCCGCUCCA 
                 329 
               
               
                   
                 UCCAGACGCUGAGGA 
                   
               
               
                   
                 GCCCCAAGGCGAGGCCCGCCGCUCC 
                 330 
               
               
                   
                 AUCCAGACGCUGAGGG 
                   
               
               
                   
               
               
                 BC 
                 AAGAUCUCGUCAUGCUUUGACGUCA 
                 331 
               
               
                   
                 AUCACCAUUGUUCCC 
                   
               
               
                   
                 AAGAUCUCGUCAUGCUUUGACGUCA 
                 332 
               
               
                   
                 AUCACCAUUGUUCC 
                   
               
               
                   
                 AAGAUCUCGUCAUGCUUUGACGCCA 
                 333 
               
               
                   
                 AUCACCAUUGUUCCC 
                   
               
               
                   
                 AAGAUCUCGUCAUGCUUUGACGUCA 
                 334 
               
               
                   
                 AUCACCAUUGUUCCA 
                   
               
               
                   
                 AAGAUCUCGUCAUGCUUUGACGUCA 
                 335 
               
               
                   
                 AUCACCAUUGUUCCU 
                   
               
               
                   
                 AAGAUCUCGUCAUGCUUUGACGUCA 
                 336 
               
               
                   
                 AUCACCAUUGUUCCCC 
                   
               
               
                   
                 AAAGAUCUCGUCAUGCUUUGACGUC 
                 337 
               
               
                   
                 AAUCACCAUUGUUCCC 
                   
               
               
                   
                 AAGAUCUCGUCAUGCCUUGACGUCA 
                 338 
               
               
                   
                 AUCACCAUUGUUCCC 
                   
               
               
                   
               
               
                 BD 
                 AUCCCCCAGGAUGAGCACGUUGCCA 
                 339 
               
               
                   
                 UGGACUGGCUAUCC 
                   
               
               
                   
                 AUCCCCAGGAUGAGCACGUUGCCAU 
                 340 
               
               
                   
                 GGACUGGCUAUCC 
                   
               
               
                   
               
               
                 BE 
                 CUGUUACAGUCUCGCGUAACCCCCC 
                 341 
               
               
                   
                 CAUCGAUGUCCUCGA 
                   
               
               
                   
                 CUGUUACAGUCUCGCGUAACCCCCC 
                 342 
               
               
                   
                 CAUCGAUGUCCUCGG 
                   
               
               
                   
                 CUGUUACAGUCUCGAGUAACCCCCC 
                 343 
               
               
                   
                 CAUCGAUGUCCUCGA 
                   
               
               
                   
                 CUGUUACAGUCUCGCGUAACCCCUC 
                 344 
               
               
                   
                 CAUCGAUGUCCUCGA 
                   
               
               
                   
                 CUGUUACAGCCUCGCGUAACCCCCC 
                 345 
               
               
                   
                 CAUCGAUGUCCUCGA 
                   
               
               
                   
                 CUGUUACAGUCUCCCGUAACCCCCC 
                 346 
               
               
                   
                 CAUCGAUGUCCUCGA 
                   
               
               
                   
               
               
                 BF 
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 347 
               
               
                   
                 ACUCCACCCUGCUCA 
                   
               
               
                   
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 348 
               
               
                   
                 ACUCCACCCUCCUCA 
                   
               
               
                   
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 349 
               
               
                   
                 ACUCCGCCCUGCUCA 
                   
               
               
                   
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 350 
               
               
                   
                 ACUCCACCCUGCU 
                   
               
               
                   
                 AGCCAGCUUUCGGCGAACCGAAUUC 
                 351 
               
               
                   
                 ACUCCACCCUGCUCA 
                   
               
               
                   
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 352 
               
               
                   
                 ACUCCACCCUGCUCG 
                   
               
               
                   
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 353 
               
               
                   
                 ACUCCACCCUGCUC 
                   
               
               
                   
                 AGCCAGCUUUCGGCAAACCGAAUUC 
                 354 
               
               
                   
                 ACUCCACCCUGCACA 
                   
               
               
                   
               
               
                 BG 
                 CACGGUAUAACCUCCUCAUAUACCU 
                 355 
               
               
                   
                 GCUGUGCCACCCGCG 
                   
               
               
                   
                 CACGGUAUAACCUCCUCAUAUACCU 
                 356 
               
               
                   
                 GCUGUGCCACCCGCA 
                   
               
               
                   
                 CACGGUAUAACCUCCUCAUAUACCU 
                 357 
               
               
                   
                 GCUGUGCCACCCACCG 
                   
               
               
                   
                 CACGGUAUAACCUCCUCAUAUACCU 
                 358 
               
               
                   
                 GCUGUGCCACCCGCU 
                   
               
               
                   
                 CACGGUAUAACCUCCUCAUAUACCU 
                 359 
               
               
                   
                 GCUGUGCCACCCACG 
                   
               
               
                   
                 CACGGUAUAACCUCCUCAUAUACCU 
                 360 
               
               
                   
                 GCUGUGCCACCCGUG 
                   
               
               
                   
                 CACGGUAUAACCUCCUCAUAUACCU 
                 361 
               
               
                   
                 GCUGUGCCGCCCGCG 
                   
               
               
                   
               
               
                 BH 
                 CCGGAAGAUCUGCUCGCACUAGCCG 
                 362 
               
               
                   
                 GAGCCCAAUCACGGC 
                   
               
               
                   
                 CCGGAAGAUCUGCUCGCACUAGUCG 
                 363 
               
               
                   
                 GAGCCCAAUCACGGC 
                   
               
               
                   
                 CCGGAGGAUCUGCUCGCACUAGCCG 
                 364 
               
               
                   
                 GAGCCCAAUCACGGC 
                   
               
               
                   
                 CCGGAAGAUCUGCUCGCAUUAGCCG 
                 365 
               
               
                   
                 GAGCCCAAUCACGGC 
                   
               
               
                   
               
               
                 BI 
                 CCUGCCGAACGGCUAAGUCGCAGCC 
                 366 
               
               
                   
                 CGACCCGCGGCAGGG 
                   
               
               
                   
                 CCUGCCGAACGGCUAAGUCGCAGCC 
                 367 
               
               
                   
                 CGACCCGCGGCAGG 
                   
               
               
                   
                 CCUGCCGAACGGCUAAGUCGCAGCC 
                 368 
               
               
                   
                 CGACCCGCGGCAGGA 
                   
               
               
                   
                 CCUGCCGAACGGCCAAGUCGCAGCC 
                 369 
               
               
                   
                 CGACCCGCGGCAGGG 
                   
               
               
                   
                 CCUGCCGAACGGCUAAGUCGCGGCC 
                 370 
               
               
                   
                 CGACCCGCGGCAGGG 
                   
               
               
                   
               
               
                 BJ 
                 CUCCGACCCGCGGACGAAGUCAACU 
                 371 
               
               
                   
                 UCCACAGUCCCACAC 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 372 
               
               
                   
                 UCCACAGUCCCACAA 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 373 
               
               
                   
                 UCCACAGUCCCACACAC 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 374 
               
               
                   
                 UCCACAGUCUCACAC 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 375 
               
               
                   
                 UCCACAGUCCCACAU 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 376 
               
               
                   
                 UCCACAGUCCCGCAC 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 377 
               
               
                   
                 UCCACGGUCCCACAC 
                   
               
               
                   
                 CUCCGACCCGCGGACGAAGUCAACU 
                 378 
               
               
                   
                 UCCACAGUCCCAUAC 
                   
               
               
                   
               
               
                 BK 
                 ACAUUAGGAUCUGCGUGAUGGGGAU 
                 379 
               
               
                   
                 CACCCGCUACAUGUC 
                   
               
               
                   
                 ACAUUUAGGAUCUGCGUGAUGGGGA 
                 380 
               
               
                   
                 UCACCCGCUACAUGUC 
                   
               
               
                   
                 GCAUUAGGAUCUGCGUGAUGGGGAU 
                 381 
               
               
                   
                 CACCCGCUACAUGUC 
                   
               
               
                   
                 ACAUUAGGAUCUGCGCGAUGGGGAU 
                 382 
               
               
                   
                 CACCCGCUACAUGUC 
                   
               
               
                   
               
               
                 BL 
                 UCUAAGAUGGGGAAGAUCUCCGGAG 
                 383 
               
               
                   
                 CACCGGGCAAUCACC 
                   
               
               
                   
                 UCUAAGAUGGGGAAGAUCUCCGGAG 
                 384 
               
               
                   
                 CACCGGGCAAUCACCC 
                   
               
               
                   
                 CCUAAGAUGGGGAAGAUCUCCGGAG 
                 385 
               
               
                   
                 CACCGGGCAAUCACC 
                   
               
               
                   
                 UCUAAGGUGGGGAAGAUCUCCGGAG 
                 386 
               
               
                   
                 CACCGGGCAAUCACC 
                   
               
               
                   
                 UCUAAGAUGGGGAAGAUCUCCGGAG 
                 387 
               
               
                   
                 CGCCGGGCAAUCACC 
                   
               
               
                   
               
               
                 BM 
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 388 
               
               
                   
                 AUCGAGAACCUAC 
                   
               
               
                   
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 389 
               
               
                   
                 CAUCAGAACCUAC 
                   
               
               
                   
                 CUACUCGAGUUCCCACGAAUCCCCC 
                 390 
               
               
                   
                 AUCGAGAACCUAC 
                   
               
               
                   
                 CUAUUCGAGUUCCCACGAAUCCCCC 
                 391 
               
               
                   
                 AUCAAGAACCUAC 
                   
               
               
                   
               
               
                 BN 
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 392 
               
               
                   
                 CCCUCACGAGAGAGG 
                   
               
               
                   
                 UGCCAAAGCCGAGGCCCGGCCAGCA 
                 393 
               
               
                   
                 UCCCUCACGAGAGAGG 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 394 
               
               
                   
                 CCCUCACGAGAGAGC 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 395 
               
               
                   
                 CCCUCACGAGAGAG 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 396 
               
               
                   
                 CCCCCACGAGAGAGG 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 397 
               
               
                   
                 CCCUCACGAGAGAGA 
                   
               
               
                   
                 UGCCAAGCCGGGGCCCGGCCAGCAU 
                 398 
               
               
                   
                 CCCUCACGAGAGAGG 
                   
               
               
                   
                 UGCCAAGCCGAGGCCCGGCCAGCAU 
                 399 
               
               
                   
                 CCCUCACGAGAGGG 
                   
               
               
                   
               
               
                 BO 
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 400 
               
               
                   
                 CCCGCCUGUGUGCUG 
                   
               
               
                   
                 CCAAGCACGUAGCCCGUGCCCCCAC 
                 401 
               
               
                   
                 CCGCCUGUGUGCUG 
                   
               
               
                   
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 402 
               
               
                   
                 CCCGCCUGUGUGCGG 
                   
               
               
                   
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 403 
               
               
                   
                 CCCACCUGUGUGCUG 
                   
               
               
                   
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 404 
               
               
                   
                 CCCGCCUGUGUGCUC 
                   
               
               
                   
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 405 
               
               
                   
                 CCCGCCUGUGUGCCG 
                   
               
               
                   
                 GCCAAAGCACGUAGCCCGUGCCCCC 
                 406 
               
               
                   
                 ACCCGCCUGUGUGCUG 
                   
               
               
                   
                 GCCAAGCACGUAGCCCGUGCCCCCA 
                 407 
               
               
                   
                 CCCGCCUGUGUGCUA 
                   
               
               
                   
               
               
                 BP 
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 408 
               
               
                   
                 AUCCAGAGUGUGAGA 
                   
               
               
                   
                 UGCCAAAGCACGAAGCCCGUGCCCC 
                 409 
               
               
                   
                 CAUCCAGAGUGUGAGA 
                   
               
               
                   
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 410 
               
               
                   
                 AUCCAGAGUGUGGGA 
                   
               
               
                   
                 UGCCAAGCACGAGGCCCGUGCCCCC 
                 411 
               
               
                   
                 AUCCAGAGUGUGAGA 
                   
               
               
                   
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 412 
               
               
                   
                 AUUCAGAGUGUGAGA 
                   
               
               
                   
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 413 
               
               
                   
                 AUCCAGAGUGCGAGA 
                   
               
               
                   
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 414 
               
               
                   
                 AUCCAGAGCGUGAGA 
                   
               
               
                   
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 415 
               
               
                   
                 AUCCAGAGUGUGAGG 
                   
               
               
                   
                 UGCCAAGCACGAAGCCCGUGCCCCC 
                 416 
               
               
                   
                 AUCCAGGGUGUGAGA 
                   
               
               
                   
               
               
                 BQ 
                 AGCCAGCUUUUGCAUACCACGUGCA 
                 417 
               
               
                   
                 AUUCACUCCACCCGUCA 
                   
               
               
                   
                 AGCCAGCUUUGCCAUACCACGUGCA 
                 418 
               
               
                   
                 AUUCACUCCACCCGUCA 
                   
               
               
                   
                 AGCCAGCCUUUGCAUACCACGUGCA 
                 419 
               
               
                   
                 AUUCACUCCACCCGUCA 
                   
               
               
                   
                 AGCCAGCUUUUGCAUACCACGUGCA 
                 420 
               
               
                   
                 AUUCACUCCACCCGUCG 
                   
               
               
                   
                 AGCCAGCUUUUGCACACCACGUGCA 
                 421 
               
               
                   
                 AUUCACUCCACCCGUCA 
                   
               
               
                   
                 AGCCAAGCUUUGCAUACCACGUGCA 
                 422 
               
               
                   
                 AUUCACUCCACCCGUCA 
                   
               
               
                   
               
               
                 BR 
                 CUUUGUAAACCCGGCAAACAAAAUC 
                 423 
               
               
                   
                 AACUUCCAUCAUCAA 
                   
               
               
                   
                 CUUUGUAAACCCGGCAAACAAAAUC 
                 424 
               
               
                   
                 AACUUCCAUCACCAA 
                   
               
               
                   
               
               
                 BS 
                 CCAUUGUAGCGACCACACAAUUCCC 
                 425 
               
               
                   
                 CAUCGGACAGCAUGG 
                   
               
               
                   
                 CCAUUGUAGCGACCACACAAUUCCC 
                 426 
               
               
                   
                 CAUCGGACAGCAUG 
                   
               
               
                   
                 CCAUUGUAGCGACCACACAAUUCCC 
                 427 
               
               
                   
                 CAUCGGACAGCGUGG 
                   
               
               
                   
                 CCAUUGUAGCGACCACACAAUUCCC 
                 428 
               
               
                   
                 CAUCGGACAGCACGG 
                   
               
               
                   
                 CCAUUGUAGCGACCACACAAUUCCC 
                 429 
               
               
                   
                 CAUCGGACAGCAUGC 
                   
               
               
                   
                 CCAUUGUAGCGACCACACAAUCCCC 
                 430 
               
               
                   
                 CAUCGGACAGCAUGG 
                   
               
               
                   
                 CCAUUGUAGCGACCACACAAUUCCC 
                 431 
               
               
                   
                 CAUCGGACAGCAUGU 
                   
               
               
                   
               
               
                 BT 
                 CUCUCGCCGUUCCCAGGCACGACAA 
                 432 
               
               
                   
                 AAUCAACUUCCCGCU 
                   
               
               
                   
                 CUCUCGCCGUUCCCAGGCGCGACAA 
                 433 
               
               
                   
                 AAUCAACUUCCCGCU 
                   
               
               
                   
                 CUCUCGCCGUUCCCGGGCACGACAA 
                 434 
               
               
                   
                 AAUCAACUUCCCGCU 
                   
               
               
                   
                 CUCUCGCCGUUCCCAGGCACGACAA 
                 435 
               
               
                   
                 AAUCAACUUCCCGCA 
                   
               
               
                   
               
               
                 BU 
                 AAGCCAAGCCGCGGCCCGGCCUUCC 
                 436 
               
               
                   
                 CAUGUGCUACUAGAG 
                   
               
               
                   
                 AAAGCCAAGCCGCGGCCCGGCCUUC 
                 437 
               
               
                   
                 CCAUGUGCUACUAGAG 
                   
               
               
                   
                 AAGCCAAAGCCGCGGCCCGGCCUUC 
                 438 
               
               
                   
                 CCAUGUGCUACUAGAG 
                   
               
               
                   
                 GAGCCAAGCCGCGGCCCGGCCUUCC 
                 439 
               
               
                   
                 CAUGUGCUACUAGAG 
                   
               
               
                   
                 AGCCAAGCCGCGGCCCGGCCUUCCC 
                 440 
               
               
                   
                 AUGUGCUACUAGAG 
                   
               
               
                   
                 AAGCCAAGCCGUGGCCCGGCCUUCC 
                 441 
               
               
                   
                 CAUGUGCUACUAGAG 
                   
               
               
                   
                 UGCCAAGCCGCGGCCCGGCCUUCCC 
                 442 
               
               
                   
                 AUGUGCUACUAGAG 
                   
               
               
                   
                 AAGCCAAGCCGAGGCCCGGCCUUCC 
                 443 
               
               
                   
                 CAUGUGCUACUAGAG 
                   
               
               
                   
               
               
                 BV 
                 CCAAAUGCCAAAGCCGUAGCCCGGC 
                 444 
               
               
                   
                 CAGUAGCCCACACGUC 
                   
               
               
                   
                 CCAAAAUGCCAAAGCCGUAGCCCGG 
                 445 
               
               
                   
                 CCAGUAGCCCACACGUC 
                   
               
               
                   
                 CCAAAUGCCAAGCCCGUAGCCCGGC 
                 446 
               
               
                   
                 CAGUAGCCCACACGUC 
                   
               
               
                   
               
               
                 BW 
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 447 
               
               
                   
                 CGUUUCCUCGUUAAG 
                   
               
               
                   
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 448 
               
               
                   
                 CGUCUCCUCGUUAAG 
                   
               
               
                   
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 449 
               
               
                   
                 CGCUUCCUCGUUAAG 
                   
               
               
                   
                 CCAUUACGCGGCGUAAUUCCCCCAU 
                 450 
               
               
                   
                 CGUUUCCUCGUUAAG 
                   
               
               
                   
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 451 
               
               
                   
                 CGUUUCCUCGUUAGG 
                   
               
               
                   
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 452 
               
               
                   
                 CGUUUCCUCGCUAAG 
                   
               
               
                   
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 453 
               
               
                   
                 CGUUUCCUCGUUAUG 
                   
               
               
                   
                 CCAUUACGCGACGUAAUUCCCCCAU 
                 454 
               
               
                   
                 CGUUUCCUCGUUAAA 
                   
               
               
                   
               
               
                 BX 
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 455 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 456 
               
               
                   
                 AGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUCCCCC 
                 457 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 458 
               
               
                   
                 CGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 459 
               
               
                   
                 GGCUCUUCCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUGAUUCCCCC 
                 460 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGUUCCCCC 
                 461 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCCCCGUAGAUUCCCC 
                 462 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
                 CCAUCUAGAUCUCCGUAGAUUCCCC 
                 463 
               
               
                   
                 GGCUCCUUCUCGC 
                   
               
               
                   
                 CCAUCUAUAUCUCCGUAGAUUCCCC 
                 464 
               
               
                   
                 GGCUCUUUCUCGC 
                   
               
               
                   
               
               
                 BY 
                 ACUGUCUGCAUACACGGUAUGCCCA 
                 465 
               
               
                   
                 ACGCCAUCCAAACCG 
                   
               
               
                   
                 ACUGUCUGCAUACACGGUAUGCCCA 
                 466 
               
               
                   
                 ACGCCAUCCAAACCGC 
                   
               
               
                   
                 ACUGUCUGCAUACAUGGUAUGCCCA 
                 467 
               
               
                   
                 ACGCCAUCCAAACCG 
                   
               
               
                   
                 ACUGUCUGCAUACACGGUAUGCCCA 
                 468 
               
               
                   
                 ACGCCAUCCAAAACCG 
                   
               
               
                   
               
               
                 BZ 
                 ACCUGCGGCUAUUGCCAGCGCCAUA 
                 469 
               
               
                   
                 AGACCCUCCACAGUA 
                   
               
               
                   
                 ACCUGCGGCUAUUGCCAGCGCCAUA 
                 470 
               
               
                   
                 AGACCCUCCACAGCA 
                   
               
               
                   
                 CCUGCGGCUAUUGCCAGCGCCAUAA 
                 471 
               
               
                   
                 GACCCUCCACAGUA 
                   
               
               
                   
                 ACCUGCGGCUAUUGCCAGCGCCAUA 
                 472 
               
               
                   
                 AGACCUUCCACAGUA 
                   
               
               
                   
                 ACCUGCGGCUAUUGCCAGCGCCAUA 
                 473 
               
               
                   
                 AGACCCUCCGCAGUA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Nucleolin Binding of Aptamer Families A-F 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Clone 
                 Kd (nM) 
                 Bmax (%) 
                 R 2   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 FAM-A 
                 10.07 
                 17.66 
                 0.9499 
               
               
                   
                 FAM-B 
                 0.8508 
                 25.2 
                 0.8335 
               
               
                   
                 FAM-C 
                 0.4285 
                 32.76 
                 0.869 
               
               
                   
                 FAM-D 
                 0.586 
                 53.6 
                 0.9447 
               
               
                   
                 FAM-E 
                 1.69 
                 23.08 
                 0.7941 
               
               
                   
                 FAM-F 
                 0.37 
                 33.6 
                 0.6520 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Nucleolin Aptamer Truncates 
               
            
           
           
               
               
               
            
               
                   
                 NCL 
                   
               
               
                   
                 Aptamer 
                 Sequence 
               
               
                   
                   
               
               
                   
                 Bv1 
                 GGAAGAGGGAUGGGUGCCAGCUUUGCAUACCAC 
               
               
                   
                   
                 GUGCAAUUCACUCCACCCGUCAC 
               
               
                   
                   
                 (SEQ ID NO: 474) 
               
               
                   
                   
               
               
                   
                 Bv2 
                 GGGAGAGAGGAAGAGGGAUGGGAGCCAGCUUUG 
               
               
                   
                   
                 CAUACCACGUGCAAUUCACUCCACCCGUCAC 
               
               
                   
                   
                 (SEQ ID NO: 475) 
               
               
                   
                   
               
               
                   
                 Dv1 
                 GGGAUGGGCACAUGGUACGCCCAAAGCGAGGCC 
               
               
                   
                   
                 CGCUGCGUAGUGCCAUAACCCAG 
               
               
                   
                   
                 (SEQ ID NO: 476) 
               
               
                   
                   
               
               
                   
                 Dv2 
                 GGGAGAGAGGAAGAGGGAUGGGCACAUGGUACG 
               
               
                   
                   
                 CCCAAAGCGAGGCCCGCUGCGUAGUGCC 
               
               
                   
                   
                 (SEQ ID NO: 477) 
               
               
                   
                   
               
               
                   
                 Ev1 
                 GGGAUGGGCACGGUCCAGCGCUAACUGUACCUG 
               
               
                   
                   
                 CUGUGCCACCCACCGCAUAACCCAGAGGUCGAU 
               
               
                   
                   
                 (SEQ ID NO: 478) 
               
               
                   
                   
               
               
                   
                 Ev2 
                 GGGAUGGGCACGGUCCAGCGCUAACUGUACCUG 
               
               
                   
                   
                 CUGUGCCACCCACCGC (SEQ ID NO: 479) 
               
               
                   
                   
               
               
                   
                 Ev3 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                   
                 GCUAACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                   
                 (SEQ ID NO: 480) 
               
               
                   
                   
               
               
                   
                 Ev4 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                   
                 GCUAACUGUACC (SEQ ID NO: 481) 
               
               
                   
                   
               
               
                   
                 Ev5 
                 GGAAGAGGGAUGGGCACGGUCCAGCGCUAACUG 
               
               
                   
                   
                 UACCUGCUGUGCCACCCACC 
               
               
                   
                   
                 (SEQ ID NO: 482) 
               
               
                   
                   
               
               
                   
                 Fv1 
                 GGGACCACGCGCCAACGUGUCAGCUACACGCCG 
               
               
                   
                   
                 UGUUCCCCGG (SEQ ID NO: 483) 
               
               
                   
                   
               
               
                   
                 Fv2 
                 GGGACCACGCGCCAACGUGUCAGCUACACGCCG 
               
               
                   
                   
                 UGUUCCCCGGCAUAACCCAGAGGUCGAU 
               
               
                   
                   
                 (SEQ ID NO: 484) 
               
               
                   
                   
               
               
                   
                 Fv3 
                 GGGAGAGAGGAAGAGGGAUGGGACCACGCGCCA 
               
               
                   
                   
                 ACGUGUCAGCUACACGCCGUGUUCCCCGG 
               
               
                   
                   
                 (SEQ ID NO: 485) 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Ev3 Truncates 
               
            
           
           
               
               
            
               
                 NCL 
                   
               
               
                 Aptamer 
                 Sequence 
               
               
                   
               
               
                 Ev3.min21 
                 GGGAUGGGCACGGUCCAGCGCUAACUGUACCUGCU 
               
               
                   
                 GUGCCACCC (SEQ ID NO: 486) 
               
               
                   
               
               
                 Ev3.min22 
                 GGGAGGAAGAGGGAUGGGCACGGUCCAGCGCUAAC 
               
               
                   
                 UGUACCUGCUGUGCCACCC (SEQ ID NO: 487) 
               
               
                   
               
               
                 Ev3.min23 
                 GGGAGGAAGAGGAUGGGCACGGUCCAGCGCUAACU 
               
               
                   
                 GUACCUGCUGUGCCACC (SEQ ID NO: 488) 
               
               
                   
               
               
                 Ev3.min24 
                 GGGAGGAAGAGGGAUGGGCACGGUCCAGCGCACUG 
               
               
                   
                 UACCUGCUGUGCCACCC (SEQ ID NO: 489) 
               
               
                   
               
               
                 Ev3.min25 
                 GGGAGGAAGAGGAUGGGCACGGUCCAGCGCACUGU 
               
               
                   
                 ACCUGCUGUGCCACC (SEQ ID NO: 490) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Additional Nucleolin Aptamers 
               
            
           
           
               
               
            
               
                 NCL 
                   
               
               
                 Aptamer 
                 Sequence 
               
               
                   
               
               
                 Cv1 
                 GGGAUGGGAAGAUCUGCUAAGUGCACGCACAAU 
               
               
                   
                 CACCAUCGAGCGUCUC 
               
               
                   
                 (SEQ ID NO: 494) 
               
               
                   
               
               
                 Cv2 
                 GGGAGAGAGGAAGAGGGAUGGGAAGAUCUGCUA 
               
               
                   
                 AGUGCACGCACAAUCACCAUCGAGCGUCUC 
               
               
                   
                 (SEQ ID NO: 495) 
               
               
                   
               
               
                 Ev6 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCUAACUGUACCUGCUGUGCC 
               
               
                   
                 (SEQ ID NO: 496) 
               
               
                   
               
               
                 Ev3min2 
                 GGGAGAGAGAGGGAUGGGCACGGUCCAGCGCUA 
               
               
                   
                 ACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 497) 
               
               
                   
               
               
                 Ev3min3 
                 GGGAGAGAGGAAGAGGAUGGGCACGGUCCAGCG 
               
               
                   
                 CUAACUGUACCUGCUGUGCCACCACCG 
               
               
                   
                 (SEQ ID NO: 498) 
               
               
                   
               
               
                 Ev3min4 
                 GGGAGAGAGGAAGAGGGAGGGCACGGUCCAGCG 
               
               
                   
                 CUAACUGUACCUGCUGUGCCCCCACCG 
               
               
                   
                 (SEQ ID NO: 499) 
               
               
                   
               
               
                 Ev3min5 
                 GGGAGAGAGGAAGAGGGAUGGGUCCAGCGCUAA 
               
               
                   
                 CUGUACCUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 500) 
               
               
                   
               
               
                 Ev3min6 
                 GGGAGAGAGGAAGAGGGAUGGGCGGUCCAGCGC 
               
               
                   
                 UAACUGUACCUGCUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 501) 
               
               
                   
               
               
                 Ev3min7 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCUAUGUCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 502) 
               
               
                   
               
               
                 Ev3min8 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCUAACUGUACCUGCUGUGCCACCC 
               
               
                   
                 (SEQ ID NO: 503) 
               
               
                   
               
               
                 Ev3min9 
                 GGGAGGAAGAGGGAUGGGCACGGUCCAGCGCUA 
               
               
                   
                 ACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 504) 
               
               
                   
               
               
                 Ev3min10 
                 GGAAGAGGGAUGGGCACGGUCCAGCGCUAACUG 
               
               
                   
                 UACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 505) 
               
               
                   
               
               
                 Ev3min11 
                 GAGAGGAAGAGGGAUGGGCACGGUCCAGCGCUA 
               
               
                   
                 ACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 506) 
               
               
                   
               
               
                 Ev3min12 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCUAACUGUACCUGCUGUGCCACCCAC 
               
               
                   
                 (SEQ ID NO: 507) 
               
               
                   
               
               
                 Ev3min13 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCUAACUGUACCUGCUGUGCCACCCCG 
               
               
                   
                 (SEQ ID NO: 508) 
               
               
                   
               
               
                 Ev3min14 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCGCG 
               
               
                   
                 CUAACUGUACCUGCUGGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 509) 
               
               
                   
               
               
                 Ev3min15 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCGCG 
               
               
                   
                 CUAACUGUACCGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 510) 
               
               
                   
               
               
                 Ev3min16 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 511) 
               
               
                   
               
               
                 Ev3min17 
                 GGGAGAGAGGAAGAGGGAUGGGCACGGUCCAGC 
               
               
                   
                 GCUAACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 512) 
               
               
                   
               
               
                 Ev3min18 
                 GGGAGAGGAAGAGGGAUGGGCACGGUCCAGCGC 
               
               
                   
                 UAACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 513) 
               
               
                   
               
               
                 Ev3min19 
                 GGGAGAGGAAGAGGGAUGGGCACGGUCCAGCGC 
               
               
                   
                 UAACUGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 514) 
               
               
                   
               
               
                 Ev3min20 
                 GAGGAAGAGGGAUGGGCACGGUCCAGCGCUAAC 
               
               
                   
                 UGUACCUGCUGUGCCACCCACCG 
               
               
                   
                 (SEQ ID NO: 515) 
               
               
                   
               
            
           
         
       
     
     Example 2—Sensitizing Cancer Cells with Nucleolin Aptamers 
     We next tested the ability of the nucleolin aptamer truncates Bvl, Ev3, Ev4, Dv2, and Fv3 to sensitize cancer cells that overexpress nucleolin on the cell surface to ionizing radiation (IR). We also included the Ev2 aptamer as a non-binding aptamer control. As shown in  FIG. 6A , Ev3 appears to be a potent radiosensitizer, significantly decreasing post-IR survival in HCT116 p53-null cells. Further radiation sensitization studies showed that Ev3 decreased post-IR survival by approximately 5-fold in HCT116 p53-null cells compared to the aptamer control Ev5, which was used as a control due to its ability to bind nucleolin protein yet lack of radiosensitizing properties ( FIG. 6B ). Given that a large number of tumors lack functional p53, which is associated with resistance to therapy, it is encouraging that the specific nucleolin aptamer Ev3 can efficiently sensitize p53-null cells to IR. 
     To determine whether the Ev3 aptamer&#39;s ability to sensitize cancer cells to ionizing radiation was specific to the nucleolin protein, we tested the aptamer on hTERT-immortalized HFF cells ( FIG. 7 ). hTERT-immortalized HFF cells that do not express nucleolin on cell surface were treated with 5 ug of indicated aptamers and exposed to 2Gy IR 48 h later. Cells were cultivated for 10 d and survival was assessed by MTT assay. As seen in  FIG. 7 , Ev3 does not sensitize HFF (human foreskin fibroblasts) that do not express nucleolin on cell surface to radiation. 
     To determine the Ev3 and Ev5 aptamers could bind nucleolin expressed on a cell surface in a concentration-dependent manner, we performed a flow cytometry analysis with HCT116 p53-/- cells. Flow cytometry analysis of MFI (mean fluorescence intensity) of DL650-labeled Ev3 and Ev5 after incubation of HCT116 p53-/- cells with indicated aptamer concentrations. As shown in  FIG. 8  and Table 9, Ev3 and Ev5 bind to nucleolin expressed on the cell surface in a concentration dependent manner. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Ev3 and Ev5 Binding Data 
               
            
           
           
               
               
               
            
               
                   
                 One site binding (hyperbola) 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Best-fit values 
                 DL650-NCL Ev3 
                 DL650-NCL Ev5 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Bmax 
                 3.214 
                 2.064 
               
               
                   
                 Kd 
                 119.2 
                 50.7 
               
               
                   
                   
               
            
           
         
       
     
     To determine whether the EV3 aptamer could be truncated without affecting its radiosensitization function, we tested some Ev3 aptamer truncates ( FIG. 10 ). HCT 116 p53 -/- colon cancer cells were treated with 5 ug of indicated full-length aptamers or Ev3 truncates and exposed to 2Gy IR 48 h later. Cells were cultivated for 10 d and survival was assessed by MTT assay.  FIG. 10  shows truncation of Ev3 resulted in reduced activity as radiosensitizer. 
     The Ev3 nucleolin aptamer has the potential for clinical application as a cancer-specific radio- and chemosensitizer and could improve the current regimens of cancer therapy. Further, the aptamer can be radiolabeled for use as a DNA damaging agent that will preferentially target tumors and simultaneously blunt the ability of the tumor cell to repair the radiation damage, thus enhancing the sensitivity of the tumor to the radioisotope. 
     Example 3—Predicted Secondary Structures for Nucleolin Aptamers 
     Predicted secondary structures for nucleolin aptamers were generated using the mfold Web Server RNA Folding Form. Predicted structures for representative aptamers from families B, C, D, E, and F are shown in  FIGS. 11A-11B, 12A-12B, 13A-13C, 14A-14D, and 15A-15B . Predicted structures for Ev3 truncates (Ev3.min2-25) are shown in  FIGS. 16-37 .