Abstract:
Specific DNA sequences for binding various clinically relevant analytes from the human body are described. Each of these sequences or their linear, two- and three-dimensional linked sequences can function in varying assay and sensor formats with varying degrees of success. Linkage of the whole or partial DNA sequences (putative binding sites) can be used to enhance specificity and affinity towards complex targets, thereby improving assay selectivity and sensitivity in many instances. In addition, a FRET-based quantitative method is described for normalizing analyte data by assessing urine creatinine and urea levels. Finally, a method is described for removing creatinine or urea by size-exclusion chromatography prior to a FRET-based aptamer assay to avoid the denaturing effects of these compounds.

Description:
CLAIM OF PRIORITY TO PRIOR APPLICATION 
       [0001]    This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 61/402,491, filed on Aug. 31, 2010; U.S. Provisional Application Ser. No. 61/463,020, filed on Feb. 10, 2011; and U.S. Non-Provisional application Ser. No. 13/199,484, filed on Aug. 31, 2011, each of which is entitled “Methods and Compositions of Nucleic Acid Ligands for Detection of Clinical Analytes Related to Human Health”, the entire disclosures of which are hereby incorporated by reference into the present disclosure, to include the Sequence Listing(s) previously submitted with the subject applications. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention pertains to the field of aptamer- and nucleic acid ligand (DNA and RNA ligand)-based diagnostics. More specifically, the application relates to single-stranded deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) ligand sequences, whether individual or linked together to form longer multiple binding site “receptors,” that specifically target and bind to clinically relevant analytes on one or more binding sites or “epitopes” from humans such as cardiovascular biomarkers, bone metabolism markers, glucose, natural or recombinant human growth hormone (“hGH” or somatotropin) and vitamins. The invention includes general DNA ligand or aptamer-based detection and quantitation of these analytes in body fluids such as blood plasma, serum, sputum or saliva, interstitial, synovial, or cerebrospinal fluid aspirates, mucus, and urine or solid biopsy samples. 
         [0004]    2. Background Information 
         [0005]    These individual or linked DNA ligand (aptamer) sequences represent valuable target analyte-responsive components of diagnostic devices or biosensors. A “biosensor” is defined as any device that employs a biologically-derived molecule as the sensing component and transduces a target analyte binding event into a detectable physical signal (including, but not limited to, changes in light intensity, absorbance, transmittance, refraction (Surface Plasmon Resonance or “SPR”), wavelength, color, agglutination of cells or particles, fluorescence intensity, fluorescence lifetime, fluorescence polarization or anisotropy, fluorescence correlation spectroscopy (“FCS”), fluorescence or Förster resonance energy transfer (“FRET”; nonradiative dipole-dipole coupling of fluorophores or fluorophores and quenchers), upconverting phosphor, two-photon interaction phenomena, Raman spectroscopy or surface-enhanced Raman spectroscopy (“SERS”), electrical conduction, electrical resistance or other electrical properties, mass, photon or radioactive particle emissions, etc). 
         [0006]    Once bonded with the target, these DNA ligand sequences can be used to qualitatively determine the presence of analyte, as well as to quantify or semi-quantify the target analyte amount in a sample using a broad variety of assay types and diagnostic or sensor platforms including, but not limited to, affinity-based lateral flow test strips, membrane blotting, SPR, surface acoustic waveguides (“SAW” devices), magnetic bead (“MB”)-based capture, plastic-adherent sandwich assays (“PASA”), chemiluminescence (“CL”), electrochemiluminescence (“ECL”), radioisotopic, fluorescence intensity, including quantum dot (“QD”) or other fluorescent nanoparticle (“FNP”) of dye-based, fluorescence lifetime, and fluorescence polarization (“FP”) assays or enzyme-linked (“ELISA-like”) microplate assays. ELISA-like assays refer to microwell or microplate assays similar to traditional Enzyme-Linked Immunosorbent Assays (“ELISA”) in which an aptamer or nucleic acid ligand is substituted for the antibody or receptor component or components, but the other components such as peroxidase or alkaline phosphatase enzymes and color-producing substrates remain the same. 
         [0007]    In addition, these DNA ligand sequences are valuable in competitive displacement assays which are not solely dependent on high affinity (strong attractive forces between a receptor and its ligand) or high avidity (high tensile or physical strength of receptor-ligand bonds) to produce sensitive detection (sub-nanoMolar or sub-nanogram levels), because the equilibrium constant (generally K a =10 6  to 10 8  to enable competition) must allow reasonable displacement of previously bound target materials to detect a change at or below nanogram or nanoMolar levels. 
         [0008]    In a competitive displacement assay, labeled DNA ligand plus labeled analyte complexes compete with unlabeled analyte to bind with the labeled DNA. After allowing the labeled and unlabeled analytes to come to equilibrium with the labeled DNA, the unlabeled target analyte may be quantitatively assayed by fluorescence intensity or other methods. Such assays would include competitive displacement FRET assays or DNA ligand “beacon” FRET assays. In a competitive displacement FRET assay the fluorophore (“F”) and quencher (“Q”) are placed in a putative binding loop or pocket so as to reside within the Förster distance of 60-85 Angstroms to enable quenching. 
         [0009]    In an aptamer beacon assay, the F and Q labels are placed on the 5′ and 3′ ends and binding of the target analyte to the beacon opens the beacon beyond the Förster quenching distance so that F is no longer quenched and emits light generally in proportion to the amount of target analyte introduced into the liquid system. Each of these types of aptamer assays and detection platforms has different applications in either central medical laboratories or in point-of-care (“POC”) sensor devices for use in emergency rooms, intensive care units, cardiac care units, or physician&#39;s offices and clinics. 
       SUMMARY OF THE INVENTION 
       [0010]    The DNA ligand sequences listed herein (Table 9) were derived by iterative cycles of affinity-based selection, washing, heated elution, and polymerase chain reaction (“PCR”) amplification of bound DNA ligands from a randomized library using immobilized target analytes for affinity selection and PCR amplification followed by cloning and Sanger dideoxynucleotide DNA sequencing. 
         [0011]    Sanger dideoxynucleotide sequencing refers to DNA chain termination due to a lack of a 3′ hydroxyl (—OH) group to link incoming bases to during DNA synthesis followed by automated fluorescence reading of the DNA sequence from an electrophoresis gel containing all of the terminated DNA fragments. DNA sequencing may be accomplished by PCR doped with dideoxynucleotides lacking hydroxyl groups at the 2′ and 3′ sugar ring positions and thereby disallowing chain formation. PCR refers to the enzymatic amplification or copying of DNA molecules with a thermo-stable DNA polymerase such as  Thermus aquaticus  polymerase (Taq) with known “primer” regions or short oligonucleotides of known sequence that can hybridize to a longer target DNA sequence to enable priming of the chain reaction (exponential doubling of the DNA target copy number with each round of amplification). 
         [0012]    A randomized library can be chemically synthesized by linking together the four deoxynucleotide triphosphate bases (adenine; A, cytosine; C, guanine; G, and thymine; T) in equal amounts (25% each), so that a combinatorial oligonucleotide arises with sequence diversity equal to 4 raised to the nth power (4 n ) where n is the desired length of the randomized region in bases. In other words, if position 1 in an oligonucleotide is allowed to consist of A, C, G, or T (diversity=4) by equal availability of all 4 bases and these 4 possibilities are multiplied by each base linking to 4 more possible bases at position 2, then this process yields 16 possible 2-base oligonucleotides (i.e., AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, TT) and so on for the entire chosen length (n) of the randomized region. This combinatorial progression displays immense diversity as a function of oligonucleotide chain length. For example, an oligonucleotide decamer of 10 base length could be expected to contain 4 n =4 10  or 1,048,576 unique DNA sequences from which to chose or select one or more DNA sequences that bind a given immobilized target analyte with the strongest affinities. 
         [0013]    The randomized oligonucleotide or DNA is designed to be flanked on either side by short primer regions of known and fixed sequences to enable PCR amplification (exponential copying) of the rare sequences that are selected from the random library by binding to the target after the non-binding members of the random library are washed away (not selected). 
         [0014]    Additional assays, such as ELISA-like plate assays or fluorescence (e.g., intensity and FRET) assays, may be used to screen or verify the value of particular DNA and RNA ligands or aptamer sequences for detection of a given target analyte in a given assay format or type of biosensor. Some of the sequences operate (bind and transduce the binding signal) more effectively in affinity-based (ELISA-like or fluorescence intensity) assays, while other DNA ligand sequences against the same targets function better in lower affinity competitive or other assays, thereby leading to more sensitive detection with lower limits of detection (sub-nanoMolar or sub-nanogram) and less cross-reactivity or more specificity for the target analyte. “Specificity” or “selectivity” means the ability to selectively exclude molecules similar in structure to the true target analyte that may interfere with the assay and give false indications of detection. All of the listed DNA ligand nucleotide sequences have potential applications in some type of assay format, because they have survived at least 5 rounds of affinity-based selection and enrichment (by PCR amplification), although some of the sequences will undoubtedly perform better in certain assay formats or configurations (in tubes, square cuvettes, membranes, or on biochips) than others. 
         [0015]    Combinations of the DNA ligands, whether in whole or in part (i.e., their binding sites of approximately 5-10 or more nucleotides or bases), could be linked together in a linear or 2-dimensional (“2-D”) or 3-dimensional (“3-D”) fashion similar to dendrimers as shown in  FIG. 1B  to bind multiple epitopes or binding sites on a complex target analyte (Ag or antigen) such as a virus or whole prokaryotic (bacterial) or eukaryotic (animal, fungal or plant) cell surface having numerous spatially separated epitopes of different types. The advantage of linking aptamers or their shorter binding pockets, loops or binding sites is that the nascent linear, 2-D, or 3-D aptamer construct will likely have improved affinity or “avidity” (tensile binding strength) making it more difficult to remove or dissociate from the target antigen. Higher affinity and avidity generally lead to greater assay sensitivity and specificity which are typically desirable traits in many assays. 
         [0016]    The linked aptamer complex will be likely to gain specificity as well since the probability of binding to multiple epitopes with any degree of success is multiplicative. Thus, the ability to bind to epitopes A, B and C equals the product of the probability of binding to A with high affinity times the probability of binding to B with high affinity times the probability of binding to C with high affinity. The product of those three fractional probabilities is clearly much less than the probability of binding to only A, B, or C independently in the absence of binding the other two or any combination of the two epitopes therein in the absence of binding the third. Hence by linking two or more aptamers or their binding regions with or without DNA or other “spacer” regions, the selectivity or specificity of aptamers or DNA ligands can be increased. 
         [0017]    This approach to binding site linkage emulates the nature of antibodies which demonstrates linkage of their “hypervariable” (“HV”) regions on the antigen combining sites of the immunoglobulin (“Ig”) light and heavy chains. In the HV regions, the variability of the 20 amino acid types is quite high and essentially represents a selection of one combination from a large combinatorial library in the protein realm (similar to down selection of a few candidate aptamers from a large diverse starting library). The trait of HV region linkage contributes to Ig affinity, avidity and specificity. Similarly, linking aptamers or aptamer binding sites for various epitopes in one, two or three dimensions will enhance larger aptamer or DNA ligand construct affinity, avidity, and selectivity or specificity as illustrated in  FIGS. 1A and 1B . 
         [0018]    The present invention provides specific DNA sequence information for nucleic acid ligands (aptamers) or their linked constructs selected from randomized pools to bind clinically important proteins, peptides, hormones, sugars, vitamins, post-translational N-acetylglucosamine (NAG or O-GlcNAc) modifications of key proteins, etc. in a variety of assay formats and sensor or diagnostic platforms for assessment of human health status. While all of the candidate sequences have been shown to bind their cognate targets, some are shown to function more effectively in affinity-based assays versus fluorescence resonance energy transfer (FRET) or other assay formats that rely more on physical parameters other than affinity such as fluorophore-quencher proximity (i.e., the Förster distance). Therefore, all of the sequences are potentially valuable for simple qualitative detection or quantitative assays, but some may function better in terms of sensitive and specific detection than others in particular assay formats. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]      FIG. 1A  illustrates an antibody, revealing the multiple hypervariable antigen combining of binding sites on both the heavy and light chains. 
           [0020]      FIG. 1B  illustrates the concept of linking aptamers or their binding sites in a linear fashion (although 2-D and 3-D linkages are also possible) to mimic the linkage of multiple hypervariable (HV) regions in antigen combining sites of antibody chains to enhance affinity, avidity, and specificity against complex target antigens (Ag) containing two or more distinct epitopes. 
           [0021]      FIG. 2  represents secondary stem-loop structures of DNA ligands or aptamers (SEQ ID NOs 3 and 4). 
           [0022]      FIGS. 3A-3F  show how the aptamer beacons from  FIG. 2  behaved as a function of decreasing CTx peptide concentration. 
           [0023]      FIG. 4A  illustrates specificity of the 15-base CTx 2R-2h aptamer beacon for the full-length 26-amino acid (AA) version of the CTx peptide. 
           [0024]      FIG. 4B  shows the same samples evaluated by a handheld fluorometer that only reports fluorescence peak height, but confirms the level of specificity. 
           [0025]      FIG. 5  shows the relative linearity and sensitivity of the 15-base CTx 2R-2h aptamer beacon assay. 
           [0026]      FIG. 6A  illustrates the ability to normalize urine concentration readings with simple aptamer beacon fluorescence readings. 
           [0027]      FIG. 6B  shows a quantitative estimation of creatinine and urea levels in urine or serum using the aptamer beacon denaturation approach. 
           [0028]      FIG. 7A  illustrates that the 2,942 dalton (26-amino acid) CTx bone peptide can be extracted from urine to avoid the denaturing effects of creatinine and urea on the CTx 2R-2h aptamer beacon assay by use of a desalting size-exclusion polyacrylamide bead column. 
           [0029]      FIG. 7B  shows that CTx peptide extracted from human urine by means of a desalting column can still be detected to a level of at least 122 ng/ml by the CTx 2R-2h aptamer beacon. 
           [0030]      FIG. 8  illustrates the secondary stem-loop structures of several aptamers which dominated a sequenced pool of vitamin D3 aptamers (abbreviated VD3 and from the SEQ ID NOs 429-526). 
           [0031]      FIGS. 9A-9D  illustrate the FRET responses of loops A, B, and C for the aptamers shown in  FIG. 8 . 
           [0032]      FIGS. 10A-10C  reveal that the VD3 Loop C beacon appears to react equally well with (A) 1-hydroxy-vitamin D2, (B) 1-hydroxy-vitamin D3 and (C) 25-hydroxy-vitamin D3. 
           [0033]      FIG. 11  shows an assessment of the VD3 Loop C beacon. 
           [0034]      FIGS. 12A and 12B  summarize assessment of the specificity or cross-reactivity of the VD3 Loop C aptamer beacon versus a variety of potential analytes or interfering species. 
           [0035]      FIGS. 13A and 13B  show fluorometric spectra from two separate trials of a competitive displacement FRET assay. 
           [0036]      FIG. 14  shows the same samples from  FIG. 13  assessed for fluorescence peak height. 
           [0037]      FIGS. 15A-15D  show four examples of aptamers from the pool of aptamers that bind recombinant human growth hormone. 
           [0038]      FIGS. 16A-16D  show four more examples of aptamers from the pool of aptamers that bind recombinant human growth hormone. 
           [0039]      FIG. 17A  shows an experimental matrix screening scheme for capture aptamer-conjugated magnetic microbeads. 
           [0040]      FIG. 17B  shows mean bar heights of three separate measurements. 
           [0041]      FIGS. 18A and 18B  give ECL line plots for assay combinations 18 and 40 from the matrix in  FIG. 17A . 
           [0042]      FIG. 19A  shows an experimental matrix screening scheme for capture aptamer-conjugated magnetic microbeads. 
           [0043]      FIG. 19B  shows mean bar heights of three separate measurements. 
           [0044]      FIG. 20A  shows the linear ECL response for the sandwich assay of  FIG. 19 . 
           [0045]      FIG. 20B  shows cross-reactivity of the combination number 4 BNP assay versus other peptide or protein analytes that might be found at the 100 ng/ml level in human blood. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0046]    There is no single preferred embodiment for use of the DNA aptamer ligand sequences or linked aptamer constructs identified herein. Rather, the sequences are useful to varying extents in a variety of assay formats and sensors or diagnostic devices chosen from at least the following list: lateral flow test strips, Surface Enhanced Raman (SERS), Surface Plasmon Resonance (SPR), Surface Acoustic or Transverse Wave (SAW or STW) detection, electrical, electrochemical, colorimetric absorbance, agglutination, ELISA-like or enzyme-linked microplate assays, magnetic bead-based capture assays, ECL or other chemiluminescence assays, radioisotopic assays and a variety of fluorescence assays including, but not limited to, fluorescence intensity, fluorescence lifetime, fluorescence polarization (FP) and Fluorescence or Förster Resonance Energy Transfer (FRET) assays (both beacon and competitive FRET (Bruno et al., 2010, 2011) in round tubes, square or flat cuvettes, or immobilized on magnetic beads, other types of microbeads, or flat surfaces such as nitrocellulose, nylon, or other membranes or on glass or plastic DNA microarrays or “biochips.” 
         [0047]    While there may appear to be considerable variability among sequences that bind the same clinical analyte targets, “epitopes” and their cognate aptamer binding sites are usually quite small (e.g., 5-10 bases) and a single target may contain numerous individual binding sites or epitopes for multiple aptamer binding. In addition, however, there is often a common or consensus sequence (designated herein by slashes between clone numbers in Table 9, e.g. —aptamer clones CTx 2, 13, 19, 20, 25, 32F or R series are identical and only listed once as SEQ ID NO 3 and 4) or common segments of 5-10 or more nucleotides in a row within otherwise different aptamer sequences that can bind a specific target epitope that may dominate the other binding sites by being more physically accessible or having stronger electrostatic, hydrogen bonding, or other attractive forces (summation of van der Waals or other weak forces). Variations in nucleotide sequences around these consensus segments or common binding sequence segments may serve to modulate the binding segment&#39;s affinity or specificity or may have no effect at all. These properties must be determined by empirical comparisons. 
       DNA Ligand (Aptamer) Selection and Generation 
       [0048]    General methods for developing DNA ligands or aptamers to the immobilized proteins, peptides, or small molecules (defined as less than 1,000 daltons) are as follows. The protein, peptide or an amino-derivative of the small molecule (such as glucosamine in the case of D-glucose or dextrose) is then added to 2×10 9  tosyl-coated magnetic beads (MBs; e.g., Dynal brand from Invitrogen Corp. Carlsbad, Calif., 2. 8 micron size) for 2 hours at 37° C. The tosyl group is a “leaving” group that allows the formation of a very stable covalent bond between primary amine groups in the target protein, peptide or amino-derivatized small molecule and therefore immobilizes the target on the surfaces of the MBs so that they can be used to probe the randomized DNA library for DNA ligands. Target molecule-conjugated MBs (or target-MBs) are collected for 2 minutes in a magnetic collection device using an external magnet and the supernate is carefully withdrawn with a pipette tip. Target-MBs are then resuspended by vortexing briefly in 1× Binding Buffer (1×BB; 0.5M NaCl, 10 mM Tris-HCl, and 1 mM MgCl 2 , pH 7.5-7.6,) and washed by agitation for 5 minutes. MBs are collected and washed three times in this manner and then resuspended in 1 ml of 1×BB. 
         [0049]    MB-based DNA ligand or aptamer development is then performed using a template library sequence such as: 5′-ATCCGTCACACCTGCTCT-N 36 -TGGTGTTGGCTCCCGTAT-3′, where N 36  represents the randomized 36-base region of the DNA library (maximal sequence diversity=4 36  in theory). Primer sequences are: 5′-ATACGGGAGCCAACACCA-3′ (designated forward) and 5′-ATCCGTCACACCTGCTCT-3′ (designated reverse) to prime the template and nascent strands for PCR, respectively. The random library is reconstituted in 500 μl of sterile nuclease-free water and heated to 95° C. for 5 minutes to ensure that the DNA library is completely single-stranded and linear. The hot DNA library solution is added to 100 μl of target-MBs (2×10 8  beads) with 600 μl of sterile 2× Binding Buffer (2×BB). The DNA library and target-MB suspension (1.2 ml) is mixed at room temperature (RT, approximately 25° C.) for 1 hour. Target-MBs with any bound DNA (round 1 aptamers) are magnetically collected. The DNA-target-MB complexes are washed three times in 400 μl of sterile 1×BB. Following the third wash, the DNA-target-MB pellet (about 75 μl) is used in a PCR reaction to amplify the bound DNA as follows. The MB pellet is split into 15 μl aliquots and added to five pre-made PCR tubes which contain most of the nonperishable ingredients of a PCR reaction beneath a wax seal. A total of 3 μl of 1:10 primer mix (10% forward primer plus 10% reverse primer) in nuclease-free deionized water or ˜20 nanomoles of each primer per ml plus 1 μl (5 U) of Taq DNA polymerase and 5 μl of 2 mM MgCl 2  are added to each of the five tubes. PCR reactions are supplemented with 0.5 μl of  E. coli  single-strand binding protein (SSBP, Stratagene Inc., La Jolla, Calif.) to inhibit high molecular weight concatamer (end to end aggregates of the DNA ligands) formation. PCR is carried out as follows: an initial 95° C. phase for 5 minutes, followed by 20 cycles of 1 minute at 95° C., 1 minute at 53° C., and 1 minute at 72° C. followed by a 72° C. completion stage for 7 minute, and refrigeration at 4° C. This constitutes the first of multiple rounds of MB-aptamer development. Iterations of the MB-aptamer development process are repeated until the desired affinity or assay sensitivity and specificity are achieved. Typically, 5-10 rounds of the MB-aptamer development process are required to achieve low ng/ml detection of target analytes. To begin the second round and all subsequent rounds, 4 complete tubes of the 5 original PCR tubes are heated to 95° C. for 5 minutes to release bound DNA from the target-MBs. The fifth tube is always retained and refrigerated as a back-up for that round of the aptamer generation process. All available DNA (25 μl per tube) is siphoned out of the hot tubes without removing the target-MBs before the tubes cool significantly and the DNA is pooled. The 100 μl of hot DNA is added to 100 μl of fresh target-MBs in 200 μl of 2×BB and allowed to mix for 1 hr at RT. Thereafter, the selection and amplification process are repeated for 3-8 more rounds with checking for 72 bp aptamer PCR products by ethidium bromide-stained 2% agarose electrophoresis after each round. Following the last round of aptamer development, aptamers are cloned into chemically competent  E. coli  using a cloning kit from Lucigen Corp. (Middleton, Wis.) and clones are sent to Sequetech, Inc. (Mountain View, Calif.) for DNA sequencing. 
       Screening of Aptamers for Highest Affinity, Lowest Cross-Reactivity and to Determine Lower Limit of Detection by Target Titration in ELISA-Like Plate Assay (“ELASA”) 
       [0050]    To evaluate, screen, and rank aptamers based on affinity against clinically relevant targets, an enzyme-linked plate assay is conducted by first immobilizing 100 μl of 1:10 diluted target (about 0.1 mg of total protein, peptide or small molecule) in 0.1M NaHCO 3  (pH 8.5) overnight at 4° C. in a covered polystyrene 96-well plate. The plate is decanted and washed three times in 250 μl of 1×BB. Each of the different 5′-biotinylated aptamers raised against the target is dissolved in 1×BB at 1.00 nmoles to 4.50 nmoles per 100 microliters and applied to their corresponding plate wells for 1 hour at room temperature (RT; ˜25° C.) with gentle mixing on an orbital shaker. The plate is decanted and washed three times in 250 μl of 1×BB for at least 5 minutes per wash with gentle mixing. One hundred μl of a 1:2,000 dilution of streptavidin-peroxidase from a 5 mg/ml stock solution in 1×BB is added per well for 30 minutes at RT with gentle mixing. The plate is decanted and washed three times with 250 μl of 1×BB per well as before. One hundred μl of ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) substrate with stabilized hydrogen peroxide (Kirkegaard Perry Laboratories, Inc., Gaithersburg, Md.) is added per well for 10 minute at RT. Finally absorbance is quantified using a microplate reader with 405 nm optical filter. 
         [0051]    As Tables 1-8 illustrate for several cardiovascular biomarker targets (Brain Natriuretic Peptide; BNP, D-Dimer; DD, Creatine Kinase-MB types I and II; MBI and MB2, Interleukin-18; IL18, and Troponin-T; Tpn all at (1 μg/ml) the initial ELASA screening is useful for ranking the relative affinity of aptamers for their respective targets by simple ranking of absorbance values at 405 nm from highest to lowest. Each of the Tables (1-8) illustrates general consistency between ELASA trials as well (i.e., the highest affinity aptamers consistently rank among the highest absorbance values between ELASA trials or plates 1-4 maximally). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for 
               
               
                 Brain Natriuretic Peptide (BNP) 
               
             
          
           
               
                   
                 Well 
                 Aptamer 
                 A 405 nm 
               
               
                   
                   
               
             
          
           
               
                 Plate 1 
               
             
          
           
               
                   
                 A12 
                 BNP - 6R 
                 2.766 
               
               
                   
                 C1 
                 BNP - 14bF 
                 2.283 
               
               
                   
                 A3 
                 BNP - 2F 
                 2.276 
               
               
                   
                 B1 
                 BNP - 7F 
                 2.227 
               
               
                   
                 E3 
                 BNP - 25cf 
                 2.215 
               
               
                   
                 A1 
                 BNP - 1F 
                 2.184 
               
               
                   
                 B4 
                 BNP - 8R 
                 2.176 
               
               
                   
                 A8 
                 BNP - 4/9R 
                 2.163 
               
               
                   
                 C3 
                 BNP - 15aF 
                 2.162 
               
               
                   
                 D4 
                 BNP - 21bR 
                 2.149 
               
             
          
           
               
                 Plate 2 
               
             
          
           
               
                   
                 A12 
                 BNP - 6R 
                 2.171 
               
               
                   
                 A3 
                 BNP - 2F 
                 2.110 
               
               
                   
                 C1 
                 BNP - 14bF 
                 2.089 
               
               
                   
                 A8 
                 BNP - 4/9R 
                 1.989 
               
               
                   
                 A1 
                 BNP - 1F 
                 1.987 
               
               
                   
                 B9 
                 BNP - 13F 
                 1.986 
               
               
                   
                 A9 
                 BNP - 5/11/15b/19/25bF 
                 1.977 
               
               
                   
                 C2 
                 BNP - 14bR 
                 1.971 
               
               
                   
                 D9 
                 BNP - 23bF 
                 1.961 
               
               
                   
                 B4 
                 BNP - 8R 
                 1.953 
               
             
          
           
               
                 Plate 3 
               
             
          
           
               
                   
                 A12 
                 BNP - 6R 
                 2.285 
               
               
                   
                 D5 
                 BNP - 22F 
                 2.272 
               
               
                   
                 E3 
                 BNP - 25cF 
                 2.240 
               
               
                   
                 D4 
                 BNP - 21bR 
                 2.212 
               
               
                   
                 C1 
                 BNP - 14bF 
                 2.206 
               
               
                   
                 B4 
                 BNP - 8R 
                 2.197 
               
               
                   
                 A9 
                 BNP - 5/11/15b/19/25bF 
                 2.196 
               
               
                   
                 A3 
                 BNP - 2F 
                 2.176 
               
               
                   
                 C3 
                 BNP - 15aF 
                 2.165 
               
               
                   
                 B5 
                 BNP - 10F (70) 
                 2.157 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for D-Dimer (DD) 
               
             
          
           
               
                   
                 Well 
                 Aptamer 
                 A 405 nm 
               
               
                   
                   
               
             
          
           
               
                 Plate 1 
               
             
          
           
               
                   
                 A10 
                 DD - 5R 
                 1.888 
               
               
                   
                 B10 
                 DD - 12R 
                 1.427 
               
               
                   
                 C7 
                 DD - 17F (71) 
                 1.261 
               
               
                   
                 B8 
                 DD - 11R (71) 
                 1.122 
               
               
                   
                 B12 
                 DD - 13R (71) 
                 1.088 
               
               
                   
                 B1 
                 DD - 7F (71) 
                 1.027 
               
               
                   
                 B7 
                 DD - 11F (71) 
                 1.021 
               
               
                   
                 B2 
                 DD - 7R (71) 
                 1.017 
               
               
                   
                 A12 
                 DD - 6R (71) 
                 0.989 
               
               
                   
                 C12 
                 DD - 20R (71) 
                 0.952 
               
             
          
           
               
                 Plate 2 
               
             
          
           
               
                   
                 A10 
                 DD - 5R 
                 1.809 
               
               
                   
                 A4 
                 DD - 2R (71) 
                 1.620 
               
               
                   
                 C7 
                 DD - 17F (71) 
                 1.237 
               
               
                   
                 B10 
                 DD - 12R 
                 1.213 
               
               
                   
                 A12 
                 DD - 6R (71) 
                 1.005 
               
               
                   
                 B7 
                 DD - 11F (71) 
                 1.003 
               
               
                   
                 B8 
                 DD - 11R (71) 
                 0.986 
               
               
                   
                 B1 
                 DD - 7F (71) 
                 0.964 
               
               
                   
                 C11 
                 DD - 20F (71) 
                 0.932 
               
               
                   
                 B6 
                 DD - 10R (71) 
                 0.930 
               
             
          
           
               
                 Plate 3 
               
             
          
           
               
                   
                 B1 
                 DD - 7F (71) 
                 0.803 
               
               
                   
                 C1 
                 DD - 14F (71) 
                 0.652 
               
               
                   
                 A10 
                 DD - 5R 
                 0.576 
               
               
                   
                 B4 
                 DD - 9R (71) 
                 0.545 
               
               
                   
                 B2 
                 DD - 7R (71) 
                 0.510 
               
               
                   
                 C4 
                 DD - 15R 
                 0.508 
               
               
                   
                 B5 
                 DD - 10F (71) 
                 0.418 
               
               
                   
                 C12 
                 DD - 20R (71) 
                 0.404 
               
               
                   
                 B10 
                 DD - 12R 
                 0.386 
               
               
                   
                 A5 
                 DD - 3F (71) 
                 0.370 
               
             
          
           
               
                 Plate 4 
               
             
          
           
               
                   
                 C1 
                 DD - 14F (71) 
                 0.816 
               
               
                   
                 B1 
                 DD - 7F (71) 
                 0.806 
               
               
                   
                 A10 
                 DD - 5R 
                 0.749 
               
               
                   
                 B10 
                 DD - 12R 
                 0.732 
               
               
                   
                 B4 
                 DD - 9R (71) 
                 0.645 
               
               
                   
                 C3 
                 DD - 15F 
                 0.635 
               
               
                   
                 B5 
                 DD - 10F (71) 
                 0.563 
               
               
                   
                 B2 
                 DD - 7R (71) 
                 0.535 
               
               
                   
                 A5 
                 DD - 3F (71) 
                 0.526 
               
               
                   
                 C4 
                 DD - 15R 
                 0.512 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for Creatine 
               
               
                 Kinase-MB Type I (MBI) 
               
             
          
           
               
                   
                 Well 
                 Aptamer 
                 A 405 nm 
               
               
                   
                   
               
             
          
           
               
                 Plate 1 
               
             
          
           
               
                   
                 F1 
                 MBI - 20F 
                 2.917 
               
               
                   
                 D2 
                 MBI - 1/4/5/8/9/17R (71) 
                 2.725 
               
               
                   
                 D6 
                 MBI - 3R (70) 
                 2.668 
               
               
                   
                 E10 
                 MBI - 16R 
                 2.665 
               
               
                   
                 E1 
                 MBI - 11F 
                 2.658 
               
               
                   
                 E2 
                 MBI - 11R 
                 2.651 
               
               
                   
                 E11 
                 MBI - 19F (69) 
                 2.589 
               
               
                   
                 E7 
                 MBI - 15F 
                 2.578 
               
               
                   
                 D1 
                 MBI - 1/4/5/8/9/17F (71) 
                 2.556 
               
               
                   
                 D4 
                 MBI - 2R (71) 
                 2.522 
               
             
          
           
               
                 Plate 2 
               
             
          
           
               
                   
                 F1 
                 MBI - 20F 
                 2.692 
               
               
                   
                 D2 
                 MBI - 1/4/5/8/9/17R (71) 
                 2.680 
               
               
                   
                 D6 
                 MBI - 3R (70) 
                 2.647 
               
               
                   
                 E1 
                 MBI - 11F 
                 2.612 
               
               
                   
                 E10 
                 MBI - 16R 
                 2.612 
               
               
                   
                 D3 
                 MBI - 2F (71) 
                 2.511 
               
               
                   
                 E2 
                 MBI - 11R 
                 2.499 
               
               
                   
                 D1 
                 MBI - 1/4/5/8/9/17F (71) 
                 2.497 
               
               
                   
                 F2 
                 MBI - 20R 
                 2.424 
               
               
                   
                 D4 
                 MBI - 2R (71) 
                 2.419 
               
             
          
           
               
                 Plate 3 
               
             
          
           
               
                   
                 F1 
                 MBI - 20F 
                 1.863 
               
               
                   
                 D2 
                 MBI - 1/4/5/8/9/17R (71) 
                 1.858 
               
               
                   
                 D3 
                 MBI - 2F (71) 
                 1.808 
               
               
                   
                 E3 
                 MBI - 12F (70) 
                 1.753 
               
               
                   
                 E2 
                 MBI - 11R 
                 1.716 
               
               
                   
                 E5 
                 MBI - 14/18F 
                 1.692 
               
               
                   
                 D4 
                 MBI - 2R (71) 
                 1.663 
               
               
                   
                 E4 
                 MBI - 12R (70) 
                 1.642 
               
               
                   
                 E1 
                 MBI - 11F 
                 1.640 
               
               
                   
                 F2 
                 MBI - 20R 
                 1.619 
               
             
          
           
               
                 Plate 4 
               
             
          
           
               
                   
                 F1 
                 MBI - 20F 
                 1.948 
               
               
                   
                 D3 
                 MBI - 2F (71) 
                 1.823 
               
               
                   
                 D6 
                 MBI - 3R (70) 
                 1.746 
               
               
                   
                 D2 
                 MBI - 1/4/5/8/9/17R (71) 
                 1.744 
               
               
                   
                 F2 
                 MBI - 20R 
                 1.742 
               
               
                   
                 D1 
                 MBI - 1/4/5/8/9/17F (71) 
                 1.733 
               
               
                   
                 E2 
                 MBI - 11R 
                 1.659 
               
               
                   
                 E3 
                 MBI - 12F (70) 
                 1.647 
               
               
                   
                 D5 
                 MBI - 3F (70) 
                 1.629 
               
               
                   
                 D4 
                 MBI - 2R (71) 
                 1.607 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for 
               
               
                 Creatine Kinase-MB Type II (MBII) 
               
             
          
           
               
                   
                 Well 
                 Aptamer 
                 A 405 nm 
               
               
                   
                   
               
             
          
           
               
                 Plate 1 
               
             
          
           
               
                   
                 G1 
                 MBII - 6/8F 
                 2.206 
               
               
                   
                 G12 
                 MBII - 12R (71) 
                 2.191 
               
               
                   
                 F12 
                 MBII - 5R 
                 2.173 
               
               
                   
                 H6 
                 MBII - 14R 
                 1.989 
               
               
                   
                 G4 
                 MBII - 7R 
                 1.910 
               
               
                   
                 H7 
                 MBII - 15F (71) 
                 1.838 
               
               
                   
                 G11 
                 MBII - 12F (71) 
                 1.827 
               
               
                   
                 H5 
                 MBII - 14F 
                 1.794 
               
               
                   
                 F10 
                 MBII - 4R (71) 
                 1.751 
               
               
                   
                 G9 
                 MBII - 11F 
                 1.732 
               
             
          
           
               
                 Plate 2 
               
             
          
           
               
                   
                 G1 
                 MBII - 6/8F 
                 2.120 
               
               
                   
                 G4 
                 MBII - 7R 
                 1.931 
               
               
                   
                 G12 
                 MBII - 12R (71) 
                 1.850 
               
               
                   
                 H5 
                 MBII - 14F 
                 1.688 
               
               
                   
                 F7 
                 MBII - 3F (71) 
                 1.682 
               
               
                   
                 G5 
                 MBII - 9F 
                 1.675 
               
               
                   
                 G11 
                 MBII - 12F (71) 
                 1.640 
               
               
                   
                 H7 
                 MBII - 15F (71) 
                 1.611 
               
               
                   
                 F12 
                 MBII - 5R 
                 1.588 
               
               
                   
                 F9 
                 MBII - 4F (71) 
                 1.586 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for Interleukin-18 (IL18) 
               
             
          
           
               
                   
                 Aptamer 
                 Well 
                 A 405 nm 
               
               
                   
                   
               
             
          
           
               
                 Plate 1 
               
             
          
           
               
                   
                 IL18 - 29F 
                 D1 
                 1.433 
               
               
                   
                 IL18 - 10F 
                 B1 
                 1.428 
               
               
                   
                 IL18 - 5F 
                 A7 
                 1.393 
               
               
                   
                 IL18 - 22R 
                 C4 
                 1.373 
               
               
                   
                 IL18 - 21R 
                 C2 
                 1.367 
               
               
                   
                 IL18 - 21F 
                 C1 
                 1.270 
               
               
                   
                 IL18 - 29R 
                 D2 
                 1.219 
               
               
                   
                 IL18 - 3F 
                 A3 
                 1.217 
               
               
                   
                 IL18 - 15R 
                 B6 
                 1.209 
               
               
                   
                 IL18 - 22F 
                 C3 
                 1.204 
               
             
          
           
               
                 Plate 2 
               
             
          
           
               
                   
                 IL18 - 22R 
                 C4 
                 1.738 
               
               
                   
                 IL18 - 21R 
                 C2 
                 1.697 
               
               
                   
                 IL18 - 10F 
                 B1 
                 1.673 
               
               
                   
                 IL18 - 29F 
                 D1 
                 1.655 
               
               
                   
                 IL18 - 5F 
                 A7 
                 1.596 
               
               
                   
                 IL18 - 22F 
                 C3 
                 1.594 
               
               
                   
                 IL18 - 26F (71) 
                 C7 
                 1.553 
               
               
                   
                 IL18 - 21F 
                 C1 
                 1.534 
               
               
                   
                 IL18 - 29R 
                 D2 
                 1.528 
               
               
                   
                 IL18 - 30F (71) 
                 D3 
                 1.525 
               
             
          
           
               
                 Plate 3 
               
             
          
           
               
                   
                 IL18 - 29F 
                 D1 
                 1.288 
               
               
                   
                 IL18 - 21R 
                 C2 
                 1.202 
               
               
                   
                 IL18 - 22R 
                 C4 
                 0.993 
               
               
                   
                 IL18 - 10F 
                 B1 
                 0.917 
               
               
                   
                 IL18 - 26F (71) 
                 C7 
                 0.886 
               
               
                   
                 IL18 - 22F 
                 C3 
                 0.855 
               
               
                   
                 IL18 - 2F 
                 A1 
                 0.844 
               
               
                   
                 IL18 - 29R 
                 D2 
                 0.795 
               
               
                   
                 IL18 - 5F 
                 A7 
                 0.791 
               
               
                   
                 IL18 - 4R 
                 A6 
                 0.785 
               
             
          
           
               
                 Plate 4 
               
             
          
           
               
                   
                 IL18 - 21R 
                 C2 
                 1.060 
               
               
                   
                 IL18 - 26F (71) 
                 C7 
                 0.841 
               
               
                   
                 IL18 - 4R 
                 A6 
                 0.835 
               
               
                   
                 IL18 - 22F 
                 C3 
                 0.677 
               
               
                   
                 IL18 - 22R 
                 C4 
                 0.662 
               
               
                   
                 IL18 - 27/31F (70) 
                 C9 
                 0.613 
               
               
                   
                 IL18 - 33R 
                 D8 
                 0.608 
               
               
                   
                 IL18 - 27/31R (70) 
                 C10 
                 0.601 
               
               
                   
                 IL18 - 5F 
                 A7 
                 0.589 
               
               
                   
                 IL18 - 29F 
                 D1 
                 0.550 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for Troponin-T (Tpn) 
               
             
          
           
               
                   
                 Aptamer 
                 Well 
                 A 405 nm 
               
               
                   
                   
               
             
          
           
               
                 Plate 1 
               
             
          
           
               
                   
                 Tpn - 20F (71) 
                 H3 
                 2.325 
               
               
                   
                 Tpn - 12R (71) 
                 G2 
                 2.070 
               
               
                   
                 Tpn - 20R (71) 
                 H4 
                 2.053 
               
               
                   
                 Tpn - 7F (71) 
                 F3 
                 2.030 
               
               
                   
                 Tpn - 18F 
                 H1 
                 1.986 
               
               
                   
                 Tpn - 18R 
                 H2 
                 1.971 
               
               
                   
                 Tpn - 6R (71) 
                 F2 
                 1.962 
               
               
                   
                 Tpn - 1R (71) 
                 E2 
                 1.949 
               
               
                   
                 Tpn - 15F (71) 
                 G7 
                 1.898 
               
               
                   
                 Tpn - 1F (71) 
                 E1 
                 1.871 
               
             
          
           
               
                 Plate 2 
               
             
          
           
               
                   
                 Tpn - 20F (71) 
                 H3 
                 2.642 
               
               
                   
                 Tpn - 20R (71) 
                 H4 
                 2.479 
               
               
                   
                 Tpn - 1F (71) 
                 E1 
                 2.374 
               
               
                   
                 Tpn - 12R (71) 
                 G2 
                 2.357 
               
               
                   
                 Tpn - 6R (71) 
                 F2 
                 2.342 
               
               
                   
                 Tpn - 18R 
                 H2 
                 2.304 
               
               
                   
                 Tpn - 7F (71) 
                 F3 
                 2.298 
               
               
                   
                 Tpn - 12F (71) 
                 G1 
                 2.252 
               
               
                   
                 Tpn - 1R (71) 
                 E2 
                 2.249 
               
               
                   
                 Tpn - 6F (71) 
                 F1 
                 2.195 
               
             
          
           
               
                 Plate 3 
               
             
          
           
               
                   
                 Tpn - 2OF (71) 
                 H3 
                 2.482 
               
               
                   
                 Tpn - 2OR (71) 
                 H4 
                 2.249 
               
               
                   
                 Tpn - 16R (71) 
                 G10 
                 1.854 
               
               
                   
                 Tpn - 16F (71) 
                 G9 
                 1.811 
               
               
                   
                 Tpn - 6R (71) 
                 F2 
                 1.656 
               
               
                   
                 Tpn - 7F (71) 
                 F3 
                 1.599 
               
               
                   
                 Tpn - 15R (71) 
                 G8 
                 1.535 
               
               
                   
                 Tpn - 9R 
                 F8 
                 1.517 
               
               
                   
                 Tpn - 1F (71) 
                 E1 
                 1.487 
               
               
                   
                 Tpn - 4bR (71) 
                 E10 
                 1.477 
               
             
          
           
               
                 Plate 4 
               
             
          
           
               
                   
                 Tpn - 20F (71) 
                 H3 
                 2.507 
               
               
                   
                 Tpn - 20R (71) 
                 H4 
                 2.259 
               
               
                   
                 Tpn - 16F (71) 
                 G9 
                 1.525 
               
               
                   
                 Tpn - 6R (71) 
                 F2 
                 1.514 
               
               
                   
                 Tpn - 16R (71) 
                 G10 
                 1.507 
               
               
                   
                 Tpn - 18F 
                 H1 
                 1.396 
               
               
                   
                 Tpn - 11F (71) 
                 F11 
                 1.386 
               
               
                   
                 Tpn - 4bR (71) 
                 E10 
                 1.350 
               
               
                   
                 Tpn - 1F (71) 
                 E1 
                 1.318 
               
               
                   
                 Tpn - 7F (71) 
                 F3 
                 1.264 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for C-Reactive Protein (CRP) 
               
             
          
           
               
                 Rank 
                 Aptamer 
                 Abs 405 nm 
               
               
                   
               
             
          
           
               
                 1 
                 CRP-5R 
                 2.332 
               
               
                 2 
                 CRP-10R 
                 2.27 
               
               
                 3 
                 CRP-2R 
                 2.210 
               
               
                 4 
                 CRP-11R 
                 2.173 
               
               
                 5 
                 CRP-17R 
                 2.165 
               
               
                 6 
                 CRP-23F 
                 2.139 
               
               
                 7 
                 CRP-10F 
                 2.100 
               
               
                 8 
                 CRP-2F 
                 2.072 
               
               
                 9 
                 CRP-9R 
                 2.066 
               
               
                 10 
                 CRP-9F 
                 2.055 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 DNA Ligand ELASA Rankings for Myoglobin (Myo) 
               
             
          
           
               
                 Rank 
                 Aptamer 
                 Abs 405 nm 
               
               
                   
               
             
          
           
               
                 1 
                 Myo-3R/4R 
                 2.395 
               
               
                 2 
                 Myo-22F 
                 2.367 
               
               
                 3 
                 Myo-15F 
                 2.344 
               
               
                 4 
                 Myo-3F/6F 
                 2.337 
               
               
                 5 
                 Myo-5R 
                 2.327 
               
               
                 6 
                 Myo-24F 
                 2.294 
               
               
                 7 
                 Myo- 
                 2.272 
               
               
                   
                 18R/27F 
               
               
                 8 
                 Myo-15R 
                 2.269 
               
               
                 9 
                 Myo-28F 
                 2.264 
               
               
                 10 
                 Myo-8F 
                 2.250 
               
               
                   
               
             
          
         
       
     
       Aptamer Beacons and Competitive FRET-Aptamer Assays 
       [0052]    Once key aptamers have been identified by the commonality of their sequences or their secondary stem-loop structures, the assay developer decides upon secondary structure loops (potential binding pockets) to label with a fluorophore (F) or quencher (Q) as illustrated by dotted lines that define the borders or limits of potential binding loops in  FIGS. 2 and 8 . Secondary stem-loop structures are easily generated by Gibbs free energy minimization with common software such as M-fold and Vienna RNA (using DNA parameters) that is freely available for public use. The researcher or inventor simply enters the DNA sequence and selects a temperature and secondary stem-loop structures like those shown in  FIG. 2  are generated. At this point, one can empirically assess candidate aptamer “beacon” potential in FRET analyte titration experiments such as those shown in  FIGS. 3A-3F . The suspected short aptamer beacon loop is re-synthesized independent of the original larger parent aptamer sequence with a fluorophore (F) such as TYE 665 attached to the 5′ end and a matched quencher (Q) such as Iowa Black attached to the 3′ end (or vice versa), purified by HPLC or other form of chromatography and assessed for fluorescence output or intensity as a function of different levels of the target analyte (e.g.,  FIGS. 3A-3F  and  FIGS. 9A-9D ). The greatest separation of fluorescence peak values or spectral emissions (e.g.,  FIGS. 3A-3F  and  FIGS. 9A-9D ) is used to define the optimal beacon since separation of fluorescence values as a function of analyte concentration is essentially the definition of a good quality fluorescence assay. 
         [0053]    Alternatively, one may label the suspected binding loops internally and place an F or a Q somewhere in the mid-section of the suspected loop other than the 3′ or 5′ end (i.e., intrachain FRET). Attachment of F or Q is usually accomplished via succinimide linkage of F- or Q-succinimides added to amino-modified aptamers at specifically chosen locations in the binding pockets. Primary amine linker moieties such as the “UniLink™” can be added internally at the time of chemical synthesis of aptamers. Typically 1 mg or more of an aptamer sequence is synthesized with a primary amine linker moiety (UniLink™) located at the approximate center of each loop structure (suspected binding pockets). Each of these internally amine-labeled aptamers is then labeled with 100 μl (0.1 mg) of F-succinimide (or alternatively Q-succinimide) for 2 hours in a 37° C. incubator, followed by purification through a 1×BB-equilibrated PD-10 (Sephadex G-25; GE Healthcare) column. In the meantime, an equal molar amount of primary amine-modified target molecule is labeled with 0.1 mg of spectrally matched Q-succinimide (to accept photons from F) at 37° C. for 2 hours and then washed three times by centrifugation at 14,000 rpm for 10 minutes per wash and resuspension in 1 ml of 1×BB. “Spectrally matched” means that most of the wavelengths of light emitted by F can be effectively absorbed by Q because its absorbance spectrum largely overlaps the emission spectrum of F. Naturally, if the aptamer is labeled with a Q-succinimide in the alternate form of the assay, the amine-modified target must be labeled with an appropriately matched F-succinimide to be quenched when bound to the Q-labeled aptamer. Pooled one ml fractions of purified F-labeled DNA aptamers are mixed with an equimolar amount of Q-labeled-amino-target analyte (or vice versa in the alternate embodiment) for 30 minutes at RT with mixing in 1×BB or phosphate buffered saline (PBS, 0.1M phosphates in 8.5 g/L sodium chloride at pH 7.2 to 7.4) and then purified through an appropriate size-exclusion chromatography column (according to molecular weight of the combined F-aptamer plus Q-target complex) to produce a purified “competitive FRET complex” consisting of F-aptamer conjugate bound to Q-labeled target. This competitive FRET complex can later be competed against unlabeled cognate analyte concentrations to increase the fluorescent light output of the liquid assay system and quantify the unlabeled analyte concentration. 
         [0054]    Generally, the aptamer beacons or competitive FRET-aptamer complexes are then diluted to a final concentration of 1-5 μg/ml in 1×BB and equally dispensed to polystyrene or methacrylate cuvettes in which 1 ml of unlabeled target at various concentrations in 1×BB, PBS or diluted blood, plasma, serum, saliva, aspirate or urine has been added already. Cuvettes are gently mixed for 15 to 20 minutes at RT prior to reading their fluorescence in the homogeneous beacon or competitive-displacement FRET assay formats using a spectrofluorometer having gratings to vary the excitation wavelength and emission scanning ability or a stationary, handheld or otherwise portable fluorometer having a more restricted or fixed excitation and emission optical filter set with a range of wavelengths for excitation and emission. 
       Aptamer or Aptamer Binding Site Linkage in One or More Dimensions 
       [0055]    The linkage of binding sites is beneficial in terms of enhancing receptor affinity, avidity (tensile binding strength), and selectivity versus complex targets with two or more distinct epitopes. This linkage can be sequential and linear (one-dimensional as in antibody heavy and light chain linkage of HV regions,  FIG. 1A ) or could be expanded into two or three dimensions much like DNA dendrimers or other more complex structures known to those skilled in the art. Linear linkage by chemical synthesis is quite facile, if one knows that aptamer DNA sequences or shorter (approximately 5-10 base) binding site sequences to be linked. One can simply design one long sequence to incorporate the desired aptamers or binding sites with repetitive poly-adenine (A), poly-cytosine (C), poly-guanine (G), poly-thymine (T), poly-uridine (U), or other intervening sequences that are unlikely to bind the target epitopes. The length of the composite aptamer construct will be limited by current chemical synthesis technology to about 200 bases. However, cellular biosynthesis or enzymatic synthesis by polymerase chain reaction (PCR) or asymmetric PCR (producing predominately single-stranded ss-DNA from a template) would not be so limited and should produce aptamer constructs up to 2,000 bases before the Taq polymerase falls off the template. The 2 kilobase Taq polymerase limit is the basis for the well-known RAPD (Random Amplification of Polymorphic DNA) method of DNA or genetic “fingerprint” analyses in which primers greater than 2 kilobases apart fail to produce a PCR product or amplicon, because Taq becomes disengaged from the template DNA before traveling 2,000 bases. In this way, lengthy aptamer constructs of less than 2 kilobases could be made from complementary DNA templates that would enable binding of different epitopes that are distal on the surface of relatively large objects such as viruses and whole bacterial or eukaryotic cells. Again, poly-A, C, G, T, or U or other linker nucleotide segments could be designed into the cDNA template to produce the resultant nascent strand to ligate aptamers or aptamer binding sites together into one contiguous linear chain with intervening linkers. 
         [0056]    For 2-D or 3-D linked aptamer structures a variety of linker chemistries are available, but the preferred embodiment is probably addition of a UniLink™ primary amine group somewhere in the mid-section of a larger multi-aptamer construct followed by covalent linkage of two or more such multi-aptamer constructs by means of bifunctional linkers such as low levels (≦1%) of glutaraldehyde, carbodiimides, sulfo-EGS, sulfo-SMCC or other such bifunctional linkers familiar to those skilled in conjugate chemistry. This strategy would result in a larger flower-like 2-D or 3-D structure consisting of two or more-lengthy multi-aptamer structures. 
         [0057]    Referring to the figures,  FIG. 1A  illustrates the general structure of an IgG antibody showing the linkage of hypervariable (HV) amino acid regions used for actual binding to target epitopes on complex antigens. Linear linkage of HV binding sites adds affinity, avidity and specificity the antibody binding to complex targets. Likewise in  FIG. 1B , aptamers or their shorter (5-10 base) binding sites can be linked during chemical or biochemical (enzymatic) synthesis to enhance aptamer binding affinity, avidity or specificity for improved assay sensitivity and selectivity. 
         [0058]      FIG. 2  is a diagram of secondary structures for two “finalists” (SEQ ID NOs 3 and 4) from the pool of 24 unique candidate aptamers (SEQ ID NOs. 1-24) that bind human Type I bone collagen C-telopeptide (CTx) indicative of bone loss when found in the urine. These 2 aptamer sequences (forward and reverse or F and R-primed) dominated the aptamer pool (12 of 38 total clones=31.6%) and were therefore considered prime candidates to investigate for possible binding pockets (secondary loop structures). These potential binding pockets are shown as loops cut off from the rest of the aptamer by dotted lines and designated according to the 12 hour clock as 2 O&#39;clock (or second hour or 2 h), 6 O&#39;clock (6 h) and 10 O&#39;clock (10 h). The overall 5′ and 3′ ends as well as a numbering system from bases 1 (at the 5′ end) to base 72 (at the 3′ end) are also indicated. A variation on the CTx 2R-2 h loop or beacon consisting of 13 and 15 bases (13b or 15b) is also indicated. The 2 O&#39;clock or second hour (2 h) aptamer was subsequently synthesized to produce an aptamer beacon (3′ end labeled with a quencher molecule and the 5′ end labeled with a fluorophore) as demarcated by the dotted lines which is capable of detecting CTx peptide to low nanogram per ml levels. 
         [0059]      FIGS. 3A-3F  show how each of the aptamer beacons derived and defined from  FIG. 2  behaved as a function of decreasing CTx peptide concentration (serial two-fold dilutions beginning with 100 micrograms per ml and ending with zero CTx peptide) in 1× binding buffer (1×BB; 0.5 M NaCl, 10 mM Tris-HCl, pH 7.5-7.6, 1 mM MgCl 2 ). In the figures “h” means hour or O&#39;clock so that 2 h refers to the 2 O&#39;clock loop from  FIG. 2 , while F means forward-primed and R means reverse-primed by reference to  FIG. 2  as well.  FIGS. 3A-3F  demonstrate that there is in fact a significant difference in the ability of each candidate loop or candidate beacon from  FIG. 2  to bind and detect a 26-amino acid (AA; full-length) CTx peptide in 1×BB. The figures show that the 15-base CTx 2R-2h loop (5′-GGTGGTGTTGGCTCC-3′) acts as a superior aptamer beacon for detection of CTx peptide, because it gives the greatest fluorescence spread as a function of CTx peptide concentration. The CTx 2R-2h loop was optimal for FRET-based detection because it gives the greatest spread or separation for various two-fold serial dilutions of the 26-AA CTx peptide beginning at 100 μg/ml. The CTx 2F-10 h loop was also noteworthy, but could not discriminate many of the different CTx concentrations. The candidate loops or beacons from this experiment were 5′ labeled with TYE665 fluorophore and 3′ labeled with Iowa Black RQ quencher and HPLC-purified prior to use at Integrated DNA Technologies, Inc. (Coralville, Iowa). Excitation on a spectrofluorometer was at 645 nm with 5 nm slits and a PMT (photomultiplier tube) setting of 900V. A slightly shorter 13-base version of the CTx-2R-2h beacon ((5′-GTGGTGTTGGCTC-3′) has also been shown to work almost as well as the 15-base version, but has slightly higher background fluorescence. Excitation in all cases was at 645±5 nm to maximally excite TYE 665 dye (fluorophore) on the 5′ end of each potential aptamer beacon with a photomultiplier tube (PMT) detector setting of 1,000 Volts. 
         [0060]      FIGS. 4A  and B demonstrate relative specificity of the CTx 2R-2h aptamer beacon.  FIG. 4A  illustrates specificity or preference of the beacon for binding to the full-length 26-AA version of the CTx peptide based on its more intense fluorescence with the full-length CTx peptide, but not with a shorter 8-amino acid segment of the same peptide using a spectrofluorometer. Since the 8-AA version of the CTx peptide (one letter amino acid sequence: EKAHDGGR) represents a subset of the larger 26-AA peptide (amino acid sequence: SAGFDFSFLPQPPQEKAHDGGRYYRA), this observation indicates where the aptamer does not bind on the larger CTx peptide and narrows down the possible binding sites to SAGFDF SFL PQPPQ or YYRA). The aptamer beacon does unexpectedly cross-react with and bind an epitope on bovine serum albumin (BSA) and since the 607 amino acid BSA protein shares its longest region of commonality with the CTx peptide at the amino acid sequence SFL or serine-phenylalanine-leucine, this may be the actual binding site of the CTx 2R-2h aptamer beacon to CTx bone peptide, although other sites cannot be ruled out completely. Again, intact bovine proteins and epitopes are not expected in human clinical samples, making the CTx 2R-2h aptamer beacon specific for its target bone peptide. 
         [0061]      FIG. 4B  shows the same samples  FIG. 4A  evaluated by a handheld fluorometer that only reports fluorescence peak height, but confirms the level of specificity seen in the spectra above. The beacon was again 5′ labeled with TYE665 fluorophore and 3′ labeled with Iowa Black quencher and HPLC-purified prior to use in assays. Excitation on the spectrofluorometer was at 645 nm with 5 nm slits and a PMT (photomultiplier tube) setting of 900V. Handheld fluorometer values were obtained with an optically modified (red light emitting diode with peak excitation at 650 nm with a 660-720 nm emission filter) Quantifluor™ device from Promega Corp. Other abbreviations used in  FIGS. 3A-3F  are: NTx; N-terminal telopeptide of human Type I bone collagen, HP; helical peptide of human bone, BSA; bovine serum albumin, DPD; deoxypyridinoline, and Pyd; pyridinoline. 
         [0062]      FIG. 5  compares the 15-base CTx 2R-2h aptamer beacon assay&#39;s titration versus different levels of the full-length CTx peptide (26 amino acids) in 1× binding buffer for 30 minutes at room temperature as assessed by the handheld Quantifluor™ from 0 to 32 ng/ml of CTx peptide with a limit of detection (LOD) of 1 ng/ml with a photodiode standard value setting of 999.0. It shows the relative linearity and sensitivity of the 15-base CTx 2R-2h aptamer beacon assay. 
         [0063]      FIGS. 6A and 6B  illustrate the linear normalization response of three different aptamer beacons raised against creatinine from the SEQ ID NOs 239-294 family to the denaturing effects of creatinine across the physiologic range found in human urine. The non-specific linear response of aptamer beacons in general to the denaturing (linearizing) effects of creatinine and urea across the physiologic range of these substances found in human urine suggest that addition of almost any aptamer beacon to urine could be used to estimate creatinine and urea levels in urine and normalize the values for other analytes in urine. It is a common practice in clinical diagnostics in which the creatinine or urea level of urine is used as a divisor for the amount of other analytes detected in urine to normalize readings between different patients. In essence, the creatinine or urea levels indicate how concentrated the urine is and that information can be used to adjust or normalize (divide by) the creatinine level to place the levels of other analytes in proper perspective (i.e., high analyte levels in a dehydrated individual may be misleading). Quantitative estimation of creatinine and urea levels in urine or serum using the aptamer beacon denaturation approach is simple (one step bind and detect), rapid (within 10-15 minutes) and facile as compared to the more complicated multi-component alkaline picrate colorimetric and time-consuming Jaffe method (i.e., results obtained greater than 35 minutes after reagent and sample preparation). 
         [0064]      FIGS. 7A and 7B  illustrate that the 2,942 dalton (26 amino acid) CTx peptide can be extracted from urine to avoid the denaturing effects of creatinine and urea on the CTx 2R-2h aptamer beacon assay (demonstrated in  FIG. 5 ) by use of a desalting size-exclusion polyacrylamide column.  FIG. 7A  shows fractions collected from a desalting column (with a 1,800 dalton molecular weight cut off) and run in an 8% polyacrylamide-sodium dodecyl sulfate (SDS) electrophoresis gel which was run against molecular weight protein standards (last lane) and Coomassie blue stained to reveal the location of the 2.9 kilodalton CTx peptide which emerged in the void volume fractions 4 and 5 (boxed). The far right lane of this gel shows molecular weight protein standards beginning at 5 kD.  FIG. 7B  shows that CTx peptide extracted from human urine by means of a desalting column can still be sensitively detected to a level of at least 122 ng/ml by the CTx 2R-2h aptamer beacon. 
         [0065]      FIG. 8  illustrates the secondary stem-loop structures of several candidate aptamers (SEQ ID NOs 433 and 434) which dominated the sequenced pool of vitamin D3 aptamers (abbreviated VD3 and from the SEQ ID NOs 429-526). Again, the lines and capital letters demarcate the loops which were considered further as binding pockets or for aptamer beacons to detect vitamin D and its isoforms or congeners. 
         [0066]      FIGS. 9A-9D  illustrate the FRET responses of loops A, B, and C for the VD3 aptamers shown in  FIG. 8  as well as handheld (Quantifluor™-P) fluorometer peak fluorescence values as a function of 25-hydroxy-vitamin D3 (calcidiol) level in 1×BB. Loop C (5′-ACTATGGT-3′) proved to be the optimal aptamer beacon based on its maximal spread or separation of fluorescence spectra as a function of vitamin D concentration as shown in the upper right quadrant after Loop C was separately synthesized with 5′-TYE 665 dye and 3′-Iowa Black quencher to convert it into a beacon for the titration experiment. The spectra were obtained by serial two-fold dilutions of calcidiol beginning at 100 μg/ml in 1×BB. The candidate VD3 loop beacons were 5′ labeled with TYE665 fluorophore and 3′ labeled with Iowa Black quencher and HPLC-purified prior to use in assays. Excitation on a spectrofluorometer was at 645 nm with 5 nm slits and a PMT setting of 900 V. Handheld fluorometer values (lower right quadrant) were obtained with an optically modified (red light emitting diode with peak excitation at 650 nm with a 660-720 nm emission filter) Quantifluor™-P device from Promega Corp. and show a lower limit of detection of about 195 ng/ml in 1×BB. 
         [0067]      FIGS. 10A-10C  show that the VD3 Loop C beacon appears to react equally well with 1-hydroxy vitamin D2, 1-hydroxy-vitamin D3 and 25-hydroxy-vitamin D3 as assessed by spectrofluorometry in 1×BB (binding buffer). Hence the Loop C beacon is only specific for the vitamin D family and cannot discriminate individual congeners. Cross-reactivity is not problematic since a number of immunoassays for vitamin D detect the total of the major forms of vitamin D2 and D3 together. Hence, the inability of the Loop C VD3 aptamer to discriminate the minor variants of vitamin D is not viewed as a limiting factor. The figures again show serial two-fold dilutions for each of the types of vitamin D beginning at 100 μg/ml in 1×BB with 1-hydroxy-vitamin D2 spectra in the top panel, 1-hydroxy-vitamin D3 spectra in the middle panel, and 25-hydroxy-vitamin D3 spectra in the bottom panel. 
         [0068]      FIG. 11  shows a graphical assessment of the VD3 Loop C beacons by the handheld fluorometer (Quantifluor™-P) in 1×BB across a range of various vitamin D concentrations and for various forms of vitamin D. The handheld reader was modified with a red-emitting (650 nm) LED light source and 660-720 nm emission filter to better work in serum where the red region optics (&gt;600 nm) can avoid much of the blue-green (&lt;600 nm) autofluorescence background of blood, serum or urine. The range shown on the x-axis spans much higher levels than previously shown. Some of the higher levels are not physiological, but might be relevant to detection of vitamin D is some vitamin-rich foods or dairy products. Lines of best fit, whether linear or exponential, are shown for the three different types of vitamin D congeners as well. The highest standard value photodetector setting of 999.0 was used in all cases. 
         [0069]      FIGS. 12A and 12B  further assess the specificity or cross-reactivity of the VD3 Loop C aptamer beacon versus a variety of potential analytes or interfering species commonly found in blood and urine by the customized handheld fluorometer ( FIG. 12A ) and by spectrofluorometry ( FIG. 12B ) H-DPD; hydroxy-deoxypyridinoline and H-Pyd; hydroxyl-pyridinoline cross linkers from bone. 
         [0070]      FIGS. 13A and 13B  show fluorometric spectra from two separate trials of a competitive displacement FRET assay using the VD3 Loop C beacon versus various levels of 25-hydroxy-vitamin D3 (two-fold serial dilutions starting at 100 μg/ml) in 1×BB. In this case, 25-hydroxy-vitamin D3 was labeled with carboxyfluorescein by Fisher esterification (reaction of a hydroxyl group on the vitamin with a carboxyl group on the fluorescein to form a covalent ester bond at acidic pH of approximately 5) followed by binding to appropriately quencher-labeled VD3 aptamer Loop C and competition against levels of calcidiol≦100 micrograms per ml). 
         [0071]      FIG. 14  shows the same samples from  FIGS. 13A and 13B  assessed for fluorescence peak height by the customized red-emitting handheld fluorometer assessed with the highest standard value photodetector setting of 999.0. The competitive FRET-aptamer assay appears to have a detection limit between 156 to 312 ng/ml in these experiments. 
         [0072]      FIGS. 15A-15D  show four examples of aptamers from the SEQ ID NOs 325-526 that bind recombinant human growth hormone (r-hGH; &gt;95% pure research grade) better than the natural form of hGH or somatotropin in ELISA-like assays to help discriminate the artificial form of hGH in potential drug or doping tests for athletes, or to monitor serum levels of exogendus doses of r-hGH administered to children with growth deficits. The ELISA-like assays were conducted with the named aptamers (SEQ ID NOs 336, 338, 343 and 345) instead of antibodies in each case and absorbances were red at 405 nm with an automated microplate reader after a 15 minute development time in the presence of ABTS substrate as is standard in the diagnostics industry. 
         [0073]      FIGS. 16A-16D  show four more examples of aptamers (from the SEQ ID NOs 325-526) that bind recombinant human growth hormone (“r-hGH”) better than the natural form of hGH or somatotropin to help discriminate the artificial form in potential drug or doping tests for atheletes and growth-challenged children. The ELISA-like assays were conducted with the named aptamers (SEQ ID NOs 348, 377, 379 and 388) instead of antibodies in each case and absorbances were red at 405 nm with an automated microplate reader after a 15 minute development time in the presence of ABTS substrate as is standard in the diagnostics industry. 
         [0074]      FIG. 17A  shows an experimental matrix screening scheme for capture aptamer-conjugated magnetic microbeads to be mixed with ruthenium trisbipyridine (Ru(bpy) 3   2+ )-labeled reporter aptamers in a sandwich assay scheme to determine which of the 7×7 top hGH and r-hGH aptamer combinations (numbered 1-49 in the scheme table) gave the strongest electrochemiluminescence (“ECL”) signal versus 10 pg/ml of r-hGH using an IGEN International Origen® ECL analyzer in phosphate buffered saline containing 0.2 M tripropylamine (“TPA”). ECL was induced by ramping the electrode voltage to 1.25 V. 
         [0075]      FIG. 17B  shows the results of this ECL matrix screening process. Mean bar heights of three separate measurements are plotted with standard deviation error bars and the best (most intense ECL) combinations are marked with asterisks. 
         [0076]      FIGS. 18A and 18B  give ECL line plots for assay combinations 18 and 40 from the matrix in  FIG. 17A  as a function of natural hGH concentrations showing sub-picogram and sub-nanogram per ml detection limits and relative linearity over the hGH ranges indicated in 50% human serum (serum diluted 1:1 in 1×BB buffer). The difference in assay regression line slope is due to doubling of the amount of aptamer-coated magnetic beads and reporter aptamer per tube in two different assay trials. 
         [0077]      FIG. 19A  shows an experimental matrix screening scheme for capture aptamer-conjugated magnetic microbeads to be mixed with ruthenium trisbipyridine (Ru(bpy) 3   2+ )-labeled reporter aptamers in a sandwich assay scheme to determine which of the 4×4 top Brain or B-type Natriuretic Peptide (“BNP”) aptamer combinations numbered 1-16 (and selected from the SEQ ID Nos. 527-562) gave the strongest ECL signal versus 100 ng of BNP using an IGEN International Origen® ECL analyzer in phosphate buffered saline containing 0.2 M TPA. 
         [0078]      FIG. 19B  shows the results of this ECL matrix screening process. Bar heights represent the means of three separate measurements plotted with standard deviation error bars and the best (most intense ECL) combinations are marked with asterisks. 
         [0079]      FIG. 20A  shows the linear ECL response for combination 4 (see  FIG. 19A ) sandwich assay versus picogram per ml levels of BNP in a 50% human serum and 1×BB diluent environment. 
         [0080]      FIG. 20B  shows cross-reactivity of the combination number 4 BNP assay versus other peptide or protein analytes that might be found at the 100 ng/ml level in human blood. 
         [0081]    Although the invention and DNA ligand (aptamer) sequences have been described with reference to specific embodiments, these descriptions are not meant to be construed in a limited sense. Various modifications of the disclosed embodiments, as well as alternative embodiments of the inventions will become apparent to persons skilled in the art upon reference to the description of the invention. It is, therefore, contemplated that the appended claims will cover such modifications that fall within the scope of the invention.