Abstract:
Methods are described for the production and use of fluorescence resonance energy transfer (FRET)-based competitive displacement aptamer assay formats. The assay schemes involve FRET in which the analyte (target) is quencher (Q)-labeled and previously bound by a fluorophore (F)-labeled aptamer such that when unlabeled analyte is added to the system and excited by specific wavelengths of light, the fluorescence intensity of the system changes in proportion to the amount of unlabeled analyte added. Alternatively, the aptamer can be Q-labeled and previously bound to an F-labeled analyte so that when unlabeled analyte enters the system, the fluorescence intensity also changes in proportion to the amount of unlabeled analyte. The F or Q is covalently linked to nucleotide triphosphates (NTPs), which are incorporated into the aptamer by various nucleic acid polymerases, such as Taq or Deep Vent Ex&amp; during PCR or asymmetric PCR, and then selected by affinity chromatography, size-exclusion, and fluorescence techniques.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of copending U.S. application Ser. No. 11/433,283 filed on May 12, 2006, which is incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to the field of aptamer- and nucleic acid-based diagnostics. More particularly, it relates to methods for the production and use of fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers for competitive displacement aptamer assay formats. The present invention provides for aptamer-related FRET assay schemes involving competitive displacement formats in which the aptamer contains fluorophores (“F”) (is F-labeled) and the target contains quenchers (“Q”) (is Q-labeled), or vice versa. The aptamer can be F-labeled or Q-labeled by incorporation of the F or Q derivatives of nucleotide triphosphates. Incorporation may be accomplished by simple chemical conjugations through bifunctional linkers, or key functional groups such as aldehydes, carbodiimides, carboxyls, N-hydroxy-succinimide (NHS) esters, thiols, etc. 
         [0004]    2. Background Information 
         [0005]    Competitive displacement aptamer FRET is a new class of assay desirable for its use in rapid (within minutes), one-step, homogeneous assays involving no wash steps (simple bind and detect quantitative assays). Others have described FRET-aptamer methods for various target analytes that consist of placing the F and Q moieties either on the 5′ and 3′ ends respectively to act like a “molecular (aptamer) beacon” or placing only F in the heart of the aptamer structure to be “quenched” by another proximal F or the DNA or RNA itself. These preceding FRET-aptamer methods are all highly engineered and based on some prior knowledge of particular aptamer sequences and secondary structures, thereby enabling clues as to where F might be placed in order to optimize FRET results. 
       SUMMARY OF THE INVENTION 
       [0006]    The nucleic acid-based “molecular beacons” snap open upon binding to an analyte or upon hybridizing to a complementary sequence, but beacons have always been end-labeled with F and Q at the 3′ and 5′ ends. The present invention provides that F-labeled or Q-labeled aptamers may be labeled anywhere in their structure that places the F or Q within the Förster distance of approximately 60-85 Angstroms of the corresponding F or Q on the labeled target analyte to achieve quenching prior to or after target analyte binding to the aptamer “binding pocket” (typically a “loop” in the secondary structure). The F and Q molecules used can include any number of appropriate fluorophores and quenchers as long as they are spectrally matched so the emission spectrum of F overlaps significantly (almost completely) with the absorption spectrum of Q. 
         [0007]    A process in which F and Q are incorporated into an aptamer population is generally referred to as “doping.” The present invention provides a new method for natural selection of F-labeled or Q-labeled aptamers that contain F-NTPs or Q-NTPs in the heart of an aptamer binding loop or pocket by PCR, asymmetric PCR (using a 100:1 forward:reverse primer ratio), or other enzymatic means. The present invention describes a strain of aptamer in which F and Q are incorporated into an aptamer population via their nucleotide triphosphate derivatives (for example, Alexfluor™-NTP ‘5, Cascade Blue®-NTP ‘5, Chromatide®-NTP&#39;s, fluorescein-NTP&#39;s, rhodamine-NTP&#39;s, Rhodamine Green™-NTP&#39;s, tetramethylrhodamine-dNTP&#39;s, Oregon Green®-NTP&#39;s, and Texas Red®-NTP&#39;s may be used to provide the fluorophores, while dabcyl-NTP&#39;s, Black Hole Quencher or BHQ™-NTP&#39;s, and QSY™ dye-NTP&#39;s may be used for the quenchers) by PCR after several rounds of selection and amplification without the F- and Q-modified bases. The advantage of this F or Q “doping” method is two-fold: 1) the method allows nature to take its course and select the most sensitive F-labeled or Q-labeled aptamer target interactions in solution, and 2) the positions of F or Q within the aptamer structure can be determined via exonuclease digestion of the F-labeled or Q-labeled aptamer followed by mass spectral analysis of the resulting fragments, thereby eliminating the need to “engineer” the F or Q moieties into a prospective aptamer binding pocket or loop. Sequence and mass spectral data can be used to further optimize the competitive aptamer FRET assay performance after natural selection as well. 
         [0008]    If the target molecule is a larger water-soluble molecule such as a protein, glycoprotein, or other water soluble macromolecule, then exposure of the nascent F-labeled and Q-labeled DNA or RNA random library to the free target analyte is done in solution. If the target is a soluble protein or other larger water-soluble molecule, then the optimal FRET-aptamer-target complexes are separated by size-exclusion chromatography. The FRET-aptamer-target complex population of molecules is the heaviest subset in solution and will emerge from a size-exclusion column first, followed by unbound FRET-aptamers and unbound proteins or other targets. Among the subset of analyte-bound aptamers there will be heterogeneity in the numbers of F- and Q-NTP&#39;s that are incorporated as well as nucleotide sequence differences, which will again effect the mass, electrical charge, and weak interaction capabilities (e.g., hydrophobicity and hydrophilicity) of each analyte-aptamer complex. These differences in physical properties of the aptamer-analyte complexes can then be used to separate out or partition the bound from unbound analyte-aptamer complexes. 
         [0009]    If the target is a small molecule (generally defined as a molecule with molecular weight of ≦1,000 Daltons), then exposure of the nascent F-labeled and Q-labeled DNA or RNA random library to the target is done by immobilizing the target. The small molecule can be immobilized on a column, membrane, plastic or glass bead, magnetic bead, quantum dot, or other matrix. If no functional group is available on the small molecule for immobilization, the target can be immobilized by the Mannich reaction (formaldehyde-based condensation reaction) on a PharmaLink™ column from Pierce Chemical Co. Elution of bound DNA from the small molecule affinity column, membrane, beads or other matrix by use of 0.2-3.0M sodium acetate at a pH of between 3 and 7. 
         [0010]    The candidate FRET-aptamers are separated based on physical properties such as charge or weak interactions by various types of HPLC, digested at each end with specific exonucleases (snake venom phosphodiesterase on the 3′ end and calf spleen phosphodiesterase on the 5′ end). The resulting oligonucleotide fragments, each one bases shorter than the predecessor, are subjected to mass spectral analysis which can reveal the nucleotide sequences as well as the positions of F and Q within the FRET-aptamers. Once the FRET-aptamer sequence is known with the positions of F and Q, it can be further manipulated during solid-phase DNA or RNA synthesis in an attempt to make the FRET assay more sensitive and specific. 
         [0011]    The competitive displacement aptamer FRET assay format of the present invention is unique. The competitive format generally requires a lower affinity aptamer in order to be able to release the F-labeled or Q-labeled target analyte and allow competition for the binding site. This may lead to less sensitivity in some assays. 
         [0012]    When running an assay, an aptamer is incorporated. In order to interact with the target molecule, the aptamer has a binding pocket or site. It is anticipated in some embodiments that the binding pocket is comprised of 3 to 6 nucleotides. These 3 or more nucleotides have a specific sequence or arrangement so as to confer the appropriate volume and conformation in 3-dimensional space to enable optimal binding to the target molecule. Where the target molecule can be any of the type described herein. 
         [0013]    The described competitive FRET aptamer uses unique aptamer sequences. The following sequences are all aptamers that bind foodborne pathogens such as  E. coli  O157:H7, Salmonella typhimurium and a surface protein from Listeria monocytogenes called “Listeriolysin.” F=forward and R=reverse primed sequences. The invention described herein may use one or more of the following aptamer sequences (the following aptamer sequences are collectively referred to as the “SEQ Aptamers.”) (The SEQ Aptamer identifiers are arranged alphabetically by aptamer target, and are listed 5′ to 3′ from left to right.): 
         [0000]    
       
         
               
             
           
               
                 Acetylcholine (ACh) Aptamer Sequences: 
               
               
                 ACh1a For 
               
               
                 ATACGGGAGCCAACACCACGATACCCGCTTATGAATTTTAAATTAATT 
               
               
                   
               
               
                 GTGATCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh1a Rev 
               
               
                 ATCCGTCACACCTGCTCTGATCACAATTAATTTAAAATTCATAAGCGG 
               
               
                   
               
               
                 GTATCGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 1b For 
               
               
                 ATACGGGAGCCAACACCAACTTTCACACATACTTGTTATACCACACGA 
               
               
                   
               
               
                 TCTTTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 1b Rev 
               
               
                 ATCCGTCACACCTGCTCTAAAAGATCGTGTGGTATAACAAGTATGTGT 
               
               
                   
               
               
                 GAAAGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 2 For 
               
               
                 ATACGGGAGCCAACACCACTTTGTAACTCATTTGTAGTTTGGGTTGCT 
               
               
                   
               
               
                 CCCCCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 2 Rev 
               
               
                 ATCCGTCACACCTGCTCTAGGGGGAGCAACCCAAACTACAAATGAGTT 
               
               
                   
               
               
                 ACAAAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 3 For 
               
               
                 ATACGGGAGCCAACACCATTTCCCGCTTATCTTCATCCACTGCTTAGC 
               
               
                   
               
               
                 ATATGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 3 Rev 
               
               
                 ATCCGTCACACCTGCTCTACATATGCTAAGCAGTGGATGAAGATAAGC 
               
               
                   
               
               
                 GGGAAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 5 For 
               
               
                 ATACGGGAGCCAACACCAGGCACTGTATCACACCGTCAAGAATGTGAT 
               
               
                   
               
               
                 CCCCTGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 5 Rev 
               
               
                 ATCCGTCACACCTGCTCTCAGGGGATCACATTCTTGACGGTGTGATAC 
               
               
                   
               
               
                 AGTGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 6 For 
               
               
                 ATACGGGAGCCAACACCATGTCATTTACCTTCATCATGACAGTGTTAG 
               
               
                   
               
               
                 TATACGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 6Rev 
               
               
                 ATCCGTCACACCTGCTCTAGGGGATCAAAGCTATGCGACCATGCGAGT 
               
               
                   
               
               
                 GGATACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 7 For 
               
               
                 ATACGGGAGCCAACACCAGTTGCCGCCTACCTTGATTATTCTACATTA 
               
               
                   
               
               
                 CCCGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 7 Rev 
               
               
                 ATCCGTCACACCTGCTCTAACGGGTAATGTAGAATAATCAAGGTAGGC 
               
               
                   
               
               
                 GGCAACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 8 For 
               
               
                 ATACGGGAGCCAACACCAGTATACATACGAAGAGTTGAAACCAATGCT 
               
               
                   
               
               
                 TCGTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 8 Rev 
               
               
                 ATCCGTCACACCTGCTCTGAACGAAGCATTGGTTTCAACTCTTCGTAT 
               
               
                   
               
               
                 GTATACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 9 For 
               
               
                 ATACGGGAGCCAACACCATACCCCGAATGGCTGTTTTCAGTACCAATA 
               
               
                   
               
               
                 TGACTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 9 Rev 
               
               
                 ATCCGTCACACCTGCTCTGAGTCATATTGGTACTGAAAACAGCCATTC 
               
               
                   
               
               
                 GGGGTATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 10 For 
               
               
                 ATACGGGAGCCAACACCACTGTCACGATCGTCGTTCCTTTTAATCCTT 
               
               
                   
               
               
                 GTGTCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 10 Rev 
               
               
                 ATCCGTCACACCTGCTCTAGACACAAGGATTAAAAGGAACGACGATCG 
               
               
                   
               
               
                 TGACAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 11 For 
               
               
                 ATACGGGAGCCAACACCACTGGACACTGACCCTCGCACTAGCTTTCTG 
               
               
                   
               
               
                 ACGGGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 11 Rev 
               
               
                 ATCCGTCACACCTGCTCTACCCGGCCGAAGAATAGTGCTCGGTACTTA 
               
               
                   
               
               
                 GTCGCGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 12 For 
               
               
                 ATACGGGAGCCAACACCATTTGGACTTTAAATAGTGGACTCCTTCTTT 
               
               
                   
               
               
                 GTCTCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 12 Rev 
               
               
                 ATCCGTCACACCTGCTCTCGAGACAAAGAAGGAGTCCACTATTTAAAG 
               
               
                   
               
               
                 TCCAAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 A25 For 
               
               
                 ATACGGGAGCCAACACCA-TCATTTGCAAATATGAATTCCACTTAAAG 
               
               
                   
               
               
                 AAATTCA-AGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 A25 Rev 
               
               
                 ATCCGTCACACCTGCTCTTGAATTTCTTTAAGTGGAATTCATATTTGC 
               
               
                   
               
               
                 AAATGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Acyl Homoserine Lactone (AHL) Quorum Sensing 
               
               
                 Molecules (N-Decanoyl-DL-Homoserine 
               
               
                 Lactone) 
               
               
                 Dec AHL 1For 
               
               
                 ATACGGGAGCCAACACCATCCTAACTGGTCTAATTTTTGCTGTTACC 
               
               
                   
               
               
                 GATCCCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Dec AHL 1 Rev 
               
               
                 ATCCGTCACTCCTGCTCTCGGGATCGGTAACAGCAAAAATTAGACCAG 
               
               
                   
               
               
                 TTAGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Dec AHL 13 For 
               
               
                 ATACGGGAGCCAACACCAGCCTGACGAAAAAATTTTATCACTAAGTGA 
               
               
                   
               
               
                 TACGCAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Dec AHL 13 Rev 
               
               
                 ATCCGTCACACCTGCTCTTGCGTATCACTTAGTGATAAAATTTTTTCG 
               
               
                   
               
               
                 TCAGGCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Dec AHL 14 For 
               
               
                 ATACGGGAGCCAACACCAGACCTACTTCAGAAACGGAAATGTTCTTAG 
               
               
                   
               
               
                 CCGTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Dec AHL 14 Rev 
               
               
                 ATCCGTCACACCTGCTCTGACGGCTAAGAACATTTCCGTTTCTGAAGT 
               
               
                   
               
               
                 AGGTCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Dec AHL 15 For 
               
               
                 ATACGGGAGCCAACACCAGGCCAACGAAACTCCTACTACATATAATGC 
               
               
                   
               
               
                 TTATGCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Dec AHL 15 Rev 
               
               
                 ATCCGTCACACCTGCTCTGCATAAGCATTATATGTAGTAGGAGTTTCG 
               
               
                   
               
               
                 TTGGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Dec AHL 17 For 
               
               
                 ATACGGGAGCCAACACCATCCTAACTGGTCTAATTTTTGCTGTTACCG 
               
               
                   
               
               
                 ATCCCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Dec AHL 17 Rev 
               
               
                 ATCCGTCACACCTGCTCTCGGGATCGGTAACAGCAAAAATTAGACCAG 
               
               
                   
               
               
                 TTAGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                   Bacillus thuringiensis  Spore Aptamer Sequence: 
               
               
                 CATCCGTCACACCTGCTCTGGCCACTAACATGGGGACCAGGTGGTGTT 
               
               
                   
               
               
                 GGCTCCCGTATC 
               
               
                   
               
               
                 Botulinum Toxin (BoNT Type A) Aptamer Sequences: 
               
               
                 BoNT A Holotoxin (Heavy Chain plus Light Chain 
               
               
                 Linked Together) 
               
               
                 CATCCGTCACACCTGCTCTGCTATCACATGCCTGCTGAAGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
               
                   
               
               
                 BoNT A 50kd Enzymatic Light Chain 
               
               
                 BoNT A Light Chain 1 
               
               
                 CATCCGTCACACCTGCTCTGGGGATGTGTGGTGTTGGCTCCCGTATCA 
               
               
                   
               
               
                 AGGGCGAATTCT 
               
               
                   
               
               
                 BoNT A Light Chain 2 
               
               
                 CATCCGTCACACCTGCTCTGATCAGGGAAGACGCCAACACGTGGTGTT 
               
               
                   
               
               
                 GGCTCCCGTATCA 
               
               
                   
               
               
                 BoNT A Light Chain 3 
               
               
                 CATCCGTCACACCTGCTCTGGGTGGTGTTGGCTCCCGTATCAAGGGCG 
               
               
                   
               
               
                 AATTCTGCAGATA 
               
               
                   
               
               
                 Campylobacter jejuni Binding Aptamers: 
               
               
                 C1 
               
               
                 CATCCGTCACACCTGCTCTGGGGAGGGTGGCGCCCGTCTCGGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATCA 
               
               
                   
               
               
                 C2 
               
               
                 CATCCGTCACACCTGCTCTGGGATAGGGTCTCGTGCTAGATGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATCA 
               
               
                   
               
               
                 C3 
               
               
                 CATCCGTCACACCTGCTCTGGACCGGCGCTTATTCCTGCTTGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATCA 
               
               
                   
               
               
                 C4 
               
               
                 CATCCGTCACACCTGCYCTGGAGCTGATATTGGATGGTCCGGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATCA 
               
               
                   
               
               
                 C5 
               
               
                 CATCCGTCACACCTGCYCYGCCCAGAGCAGGTGTGACGGATGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATCA 
               
               
                   
               
               
                 C6 
               
               
                 CATCCGTCACACCTGCYCYGCCGGACCATCCAATATCAGCTGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATCA 
               
               
                   
               
               
                 Diazinon Binding Aptamers 
               
               
                 D12 Forward 
               
               
                 ATACGGGAGCCAACACCATTAAATCAATTGTGCCGTGTTGGTCTTGTC 
               
               
                   
               
               
                 TCATCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 D12 Reverse 
               
               
                 ATCCGTCACACCTGCTCTCGATGAGACAAGACCAACACGGCACAATTG 
               
               
                   
               
               
                 ATTTAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 D17 Forward 
               
               
                 ATACGGGAGCCAACACCATTTTTATTATCGGTATGATCCTACGAGTTC 
               
               
                   
               
               
                 CTCCCAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 D17 Reverse 
               
               
                 ATCCGTCACACCTGCTCTTGGGAGGAACTCGTAGGATCATACCGATAA 
               
               
                   
               
               
                 TAAAAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 D18 Forward 
               
               
                 ATACGGGAGCCAACACCACCGTATATCTTATTATGCACAGCATCACGA 
               
               
                   
               
               
                 AAGTGCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 D18 Reverse 
               
               
                 ATCCGTCACACCTGCTCTGCACTTTCGTGATGCTGTGCATAATAAGAT 
               
               
                   
               
               
                 ATACGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 D19 Forward 
               
               
                 ATACGGGAGCCAACACCATTAACGTTAAGCGGCCTCACTTCTTTTAAT 
               
               
                   
               
               
                 CCTTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 D19 Reverse 
               
               
                 ATCCGTCACACCTGCTCTGAAAGGATTAAAAGAAGTGAGGCCGCTTAA 
               
               
                   
               
               
                 CGTTAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 D20 Forward 
               
               
                 ATCCGTCACACCTGCTCTAATATAGAGGTATTGCTCTTGGACAAGGTA 
               
               
                   
               
               
                 CAGGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 D20 Reverse 
               
               
                 ATACGGGAGCCAACACCATCCCTGTACCTTGTCCAAGAGCAATACCTC 
               
               
                   
               
               
                 TATATTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 D25 Forward 
               
               
                 ATACGGGAGCCAACACCATTAACGTTAAGCGGCCTCACTTCTTTTAAT 
               
               
                   
               
               
                 CCTTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 D25 Reverse 
               
               
                 ATCCGTCACACCTGCTCTGAAAGGATTAAAAGAAGTGAGGCCGCTTAA 
               
               
                   
               
               
                 CGTTAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Glucosamine (from LPS) Forward Aptamer Sequences: 
               
               
                 G 1 For 
               
               
                 ATCCGTCACACCTGCTCTAATTAGGATACGGGGCAACAGAACGAGAGG 
               
               
                   
               
               
                 GGGGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G 2 For 
               
               
                 ATCCGTCACACCTGCTCTCGGACCAGGTCAGACAAGCACATCGGATAT 
               
               
                   
               
               
                 CCGGCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G 4 For 
               
               
                 ATCCGTCACACCTGCTCTAATTAGGATACGGGGCAACAGAACGAGAGG 
               
               
                   
               
               
                 GGGGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G 5 For 
               
               
                 ATCCGTCACACCTGCTCTTGAGTCAAAGAGTTTAGGGAGGAGCTAACA 
               
               
                   
               
               
                 TAACAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G7 For 
               
               
                 ATCCGTCACACCTGCTCTAACAACAATGCATCAGCGGGCTGGGAACGC 
               
               
                   
               
               
                 ATGCGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G 8 For 
               
               
                 ATCCGTCACACCTGCTCTGAACAGGTTATAAGCAGGAGTGATAGTTTC 
               
               
                   
               
               
                 AGGATCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G 9 For 
               
               
                 ATCCGTCACACCTGCTCTCGGCGGCTCGCAAACCGAGTGGTCAGCACC 
               
               
                   
               
               
                 CGGGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 G 10 For 
               
               
                 ATCCGTCACACCTGCTCTGCGCAAGACGTAATCCACAAGACCGTGAAA 
               
               
                   
               
               
                 ACATAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Glucosamine (from LPS) Reverse Sequences: 
               
               
                 G 1 Rev 
               
               
                 ATACGGGAGCCAACACCATTCCCCCCTCTCGTTCTGTTGCCCCGTATC 
               
               
                   
               
               
                 CTAATTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 2 Rev 
               
               
                 ATACGGGAGCCAACACCAGCCGGATATCCGATGTGCTTGTCTGACCTG 
               
               
                   
               
               
                 GTCCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 4 Rev 
               
               
                 ATACGGGAGCCAACACCATTCCCCCCTCTCGTTCTGTTGCCCCGTATC 
               
               
                   
               
               
                 CTAATTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 5 Rev 
               
               
                 ATACGGGAGCCAACACCACTGTTATGTTAGCTCCTCCCTAAACTCTTT 
               
               
                   
               
               
                 GACTCAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 7 Rev 
               
               
                 ATACGGGAGCCAACACCACCGCATGCGTTCCCAGCCCGCTGATGCATT 
               
               
                   
               
               
                 GTTGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 8 Rev 
               
               
                 ATACGGGAGCCAACACCAGATCCTGAAACTATCACTCCTGCTTATAAC 
               
               
                   
               
               
                 CTGTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 9 Rev 
               
               
                 ATACGGGAGCCAACACCAACCCGGGTGCTGACCACTCGGTTTGCGAGC 
               
               
                   
               
               
                 CGCCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 G 10 Rev 
               
               
                 ATACGGGAGCCAACACCACTATGTTTTCACGGTCTTGTGGATTACGTC 
               
               
                   
               
               
                 TTGCGCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 KDO Antigen from LPS (Forward Primed): 
               
               
                 K 2 For 
               
               
                 ATCCGTCACACCTGCTCTAGGCGTAGTGACTAAGTCGCGCGAAAATCA 
               
               
                   
               
               
                 CAGCATTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 K 5 For 
               
               
                 ATCCGTCACACCTGCTCTCAGCGGCAGCTATACAGTGAGAACGGACTA 
               
               
                   
               
               
                 GTGCGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 K 7 For 
               
               
                 ATCCGTCACACCTGCTCTGGCAAATAATACTAGCGATGATGGATCTGG 
               
               
                   
               
               
                 ATAGACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 K 8 For 
               
               
                 ATCCGTCACACCTGCTCTGGGGGTGCGACTTAGGGTAAGTGGGAAAGA 
               
               
                   
               
               
                 CGATGCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 K 9 For 
               
               
                 ATCCGTCACACCTGCTCTCAAGAGGAGATGAACCAATCTTAGTCCGAC 
               
               
                   
               
               
                 AGGCGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 K 10 For 
               
               
                 ATCCGTCACACCTGCTCTGGCCCGGAATTGTCATGACGTCACCTACAC 
               
               
                   
               
               
                 CTCCTGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 KDO Antigen from LPS (Reverse Primed): 
               
               
                 K 2 Rev 
               
               
                 ATACGGGAGCCAACACCAATGCTGTGATTTTCGCGCGACTTAGTCACT 
               
               
                   
               
               
                 ACGCCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 K 5 Rev 
               
               
                 ATACGGGAGCCAACACCAACGCACTAGTCCGTTCTCACTGTATAGCTG 
               
               
                   
               
               
                 CCGCTGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 K 7 Rev 
               
               
                 ATACGGGAGCCAACACCAGTCTATCCAGATCCATCATCGCTAGTATTA 
               
               
                   
               
               
                 TTTGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 K 8 Rev 
               
               
                 ATACGGGAGCCAACACCAGCATCGTCTTTCCCACTTACCCTAAGTCGC 
               
               
                   
               
               
                 ACCCCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 K 9 Rev 
               
               
                 ATACGGGAGCCAACACCACCGCCTGTCGGACTAAGATTGGTTCATCTC 
               
               
                   
               
               
                 CTCTTGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 K 10 Rev 
               
               
                 ATACGGGAGCCAACACCACAGGAGGTGTAGGTGACGTCATGACAATTC 
               
               
                   
               
               
                 CGGGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   Leishmania donovani  Binding Aptamer Sequences: 
               
               
                   Leishmania donovani  Clone: 940-3 
               
               
                 Forward: 
               
               
                 GATACGGGAGCCAACACCACCCGTATCGTTCCCAATGCACTCAGAGCA 
               
               
                   
               
               
                 GGTGTGACGGATG 
               
               
                   
               
               
                 Reverse: 
               
               
                 CATCCGTCACACCTGCTCTGAGTGCATTGGGAACGATACGGGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATG 
               
               
                   
               
               
                   Leishmania donovani  Clone: 940-5 
               
               
                 Forward: 
               
               
                 GATACGGGAGCCAACACCACGTTCCCATACAAGTTACTGACAGAGCAG 
               
               
                   
               
               
                 GTGTGACGGATG 
               
               
                   
               
               
                 Reverse: 
               
               
                 CATCCGTCACACCTGCTCTGTCAGTAACTTGTATGGGAACGTGGTGTT 
               
               
                   
               
               
                 GGCTCCCGTATC 
               
               
                   
               
               
                 Whole LPS from  E. coli  O111: B4 Binding 
               
               
                 Aptamer Sequences (Forward Primed): 
               
               
                 LPS 1 For 
               
               
                 ATCCGTCACCCCTGCTCTCGTCGCTATGAAGTAACAAAGATAGGAGCA 
               
               
                   
               
               
                 ATCGGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 3 For 
               
               
                 ATCCGTCACACCTGCTCTAACGAAGACTGAAACCAAAGCAGTGACAGT 
               
               
                   
               
               
                 GCTGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 4 For 
               
               
                 ATCCGTCACACCTGCTCTCGGTGACAATAGCTCGATCAGCCCAAAGTC 
               
               
                   
               
               
                 GTCAGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 6 For 
               
               
                 ATCCGTCACACCTGCTCTAACGAAATAGACCACAAATCGATACTTTAT 
               
               
                   
               
               
                 GTTATTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 7 For 
               
               
                 ATCCGTCACACCTGCTCTGTCGAATGCTCTGCCTGGAAGAGTTGTTAG 
               
               
                   
               
               
                 CAGGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 8 For 
               
               
                 ATCCGTCACACCTGCTCTTAAGCCGAGGGGTAAATCTAGGACAGGGGT 
               
               
                   
               
               
                 CCATGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 9 For 
               
               
                 ATCCGTCACACCTGCTCTACTGGCCGGCTCAGCATGACTAAGAAGGAA 
               
               
                   
               
               
                 GTTATGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LPS 10 For 
               
               
                 ATCCGTCACACCTGCTCTGGTACGAATCACAGGGGATGCTGGAAGCTT 
               
               
                   
               
               
                 GGCTCTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Whole LPS from  E. coli  O111: B4 Binding Aptamer 
               
               
                 Sequences (Reverse Primed): 
               
               
                 LPS 1 Rev 
               
               
                 ATACGGGAGCCAACACCACCCGATTGCTCCTATCTTTGTTACTTCATA 
               
               
                   
               
               
                 GCGACGAGAGCAGGGGTGACGGAT 
               
               
                   
               
               
                 LPS 3 Rev 
               
               
                 ATACGGGAGCCAACACCATTCAGCACTGTCACTGCTTTGGTTTCAGTC 
               
               
                   
               
               
                 TTCGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 4 Rev 
               
               
                 ATACGGGAGCCAACACCATCTGACGACTTTGGGCTGATCGAGCTATT 
               
               
                   
               
               
                 GTCACCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 6 Rev 
               
               
                 ATACGGGAGCCAACACCAATAACATAAAGTATCGATTTGTGGTCTATT 
               
               
                   
               
               
                 TCGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 7 Rev 
               
               
                 ATACGGGAGCCAACACCATCCCTGCTAACAACTCTTCCAGGCAGAGCA 
               
               
                   
               
               
                 TTCGACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 8 Rev 
               
               
                 ATACGGGAGCCAACACCATCATGGACCCCTGTCCTAGATTTACCCCT 
               
               
                   
               
               
                 CGGCTTAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 9 Rev 
               
               
                 ATACGGGAGCCAACACCACATAACTTCCTTCTTAGTCATGCTGAGCC 
               
               
                   
               
               
                 GGCCAGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 10 Rev 
               
               
                 ATACGGGAGCCAACACCAAGAGCCAAGCTTCCAGCATCCCCTGTGAT 
               
               
                   
               
               
                 TCGTACCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Methylphosphonic Acid (MPA) Binding Aptamer 
               
               
                 Sequences: 
               
               
                 MPA Forward 
               
               
                 ATACGGGAGCCAACACCATTAAATCAATTGTGCCGTGTTCCTCTTGTC 
               
               
                   
               
               
                 TCATCGAGAGCAGGTTGTACGGAT 
               
               
                   
               
               
                 MPA Reverse 
               
               
                 ATCCGTACAACCTGCTCTCGATGAGACAAGAGGAACACGGCACAATTG 
               
               
                   
               
               
                 ATTTAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Malathion Binding Aptamer Sequences: 
               
               
                 M17 Forward 
               
               
                 ATACGGGAGCCAACACCAGCAGTCAAGAAGTTAAGAGAAAAACAATTG 
               
               
                   
               
               
                 TGTATAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 M17 Reverse 
               
               
                 ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTT 
               
               
                   
               
               
                 GACTGCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 M21 Forward 
               
               
                 ATCCGTCACACCTGCTCTGCGCCACAAGATTGCGGAAAGACACCCGGG 
               
               
                   
               
               
                 GGGCTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 M21 Reverse 
               
               
                 ATACGGGAGCCAACACCAAGCCCCCCGGGTGTCTTTCCGCAATCTTGT 
               
               
                   
               
               
                 GGCGCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 M25 Forward 
               
               
                 ATCCGTCACACCTGCTCTGGCCTTATGTAAAGCGTTGGGTGGTGTTGG 
               
               
                   
               
               
                 CTCCCGTAT 
               
               
                   
               
               
                 M25 Reverse 
               
               
                 ATACGGGAGCCAACACCACCCAACGCTTTACATAAGGCCAGAGCAGGT 
               
               
                   
               
               
                 GTGACGGAT 
               
               
                   
               
               
                 Poly-D-Glutamic Acid Binding Aptamer Sequences: 
               
               
                 PDGA 2F 
               
               
                 CATCCGTCACACCTGCTCTGGTTCGCCCCGGTCAAGGAGAGTGGTGTT 
               
               
                   
               
               
                 GGCTCCCGTATC 
               
               
                   
               
               
                 PDGA 2R 
               
               
                 GATACGGGAGCCAACACCACTCTCCTTGACCGGGGCGAACCAGAGCA 
               
               
                   
               
               
                 GGTGTGACGGATG 
               
               
                   
               
               
                 PDGA 5F 
               
               
                 CATCCGTCACACCTGCTCTGGATAAGATCAGCAACAAGTTAGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTATC 
               
               
                   
               
               
                 PDGA 5R 
               
               
                 GATACGGGAGCCAACACCACTAACTTGTTGCTGATCTTATCAGAGCAG 
               
               
                   
               
               
                 GTGTGACGGATG 
               
               
                   
               
               
                 Rough Ra Mutant LPS Core Antigen Binding Aptamer 
               
               
                 Sequences (Forward Primed): 
               
               
                 R 1F 
               
               
                 ATCCGTCACACCTGCTCTCCGCACGTAGGACCACTTTGGTACACGCTC 
               
               
                   
               
               
                 CCGTAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 5F 
               
               
                 ATCCGTCACACCTGCTCTACGGATGAACGAAGATTTTAAAGTCAAGCT 
               
               
                   
               
               
                 AATGCATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 6F 
               
               
                 ATCCGTCACACCTGCTCTGTAGTGAAGAGTCCGCAGTCCACGCTGTTC 
               
               
                   
               
               
                 AACTCATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 7F 
               
               
                 ATCCGTCACACCTGCTCTACCGGCTGGCACGGTTATGTGTGACGGGCG 
               
               
                   
               
               
                 AAGATATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 8F 
               
               
                 ATCCGTCACACCTGCTCTACCGGCTGGCACGGTTATGTGTGACGGGCG 
               
               
                   
               
               
                 AAGATATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 9F 
               
               
                 ATCCGTCACACCTGCTCTGCGTGTGGAGCGCCTAGGTGAGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTAT 
               
               
                   
               
               
                 R 10F 
               
               
                 ATCCGTCACACCTGCTCTGATGTCCCTTTGAAGAGTTCCATGACGCTG 
               
               
                   
               
               
                 GCTCCTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Rough Ra Mutant LPS Core Antigen Binding Aptamer 
               
               
                 Sequences (Reverse Primed): 
               
               
                 R 1R 
               
               
                 ATACGGGAGCCAACACCACTACGGGAGCGTGTACCAAAGTGGTCCTAC 
               
               
                   
               
               
                 GTGCGGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 5R 
               
               
                 ATACGGGAGCCAACACCATGCATTAGCTTGACTTTAAAATCTTCGTTC 
               
               
                   
               
               
                 ATCCGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 6R 
               
               
                 ATACGGGAGCCAACACCATGAGTTGAACAGCGTGGACTGCGGACTCTT 
               
               
                   
               
               
                 CACTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 7R 
               
               
                 ATACGGGAGCCAACACCATATCTTCGCCCGTCACACATAACCGTGCCA 
               
               
                   
               
               
                 GCCGGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 8R 
               
               
                 ATACGGGAGCCAACACCATATCTTCGCCCGTCACACATAACCGTGCCA 
               
               
                   
               
               
                 GCCGGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 9R 
               
               
                 ATACGGGAGCCAACACCACTCACCTAGGCGCTCCACACGCAGAGCAGG 
               
               
                   
               
               
                 TGTGACGGAT 
               
               
                   
               
               
                 R 10R 
               
               
                 ATACGGGAGCCAACACCAAGGAGCCAGCGTCATGGAACTCTTCAAAGG 
               
               
                   
               
               
                 GACATCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman Binding Aptamer Sequences: 
               
               
                 Soman 20F 
               
               
                 ATACGGGAGCCAACACCATAGTGTTGGGCCAATACGGTAACGTGTCCT 
               
               
                   
               
               
                 TGGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 20R 
               
               
                 ATCCGTCACACCTGCTCTCCAAGGACACGTTACCGACGAATTGGCCCA 
               
               
                   
               
               
                 ACACTATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 23F 
               
               
                 ATACGGGAGCCAACACCACACATACGAGTTATCTCGAGTAGAGCATGT 
               
               
                   
               
               
                 TTTGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 23R 
               
               
                 ATCCGTCACACCTGCTCTGGCAAAACATGCTCTACTCGAGATAACTCG 
               
               
                   
               
               
                 TATGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 24F 
               
               
                 ATACGGGAGCCAACACCAGGCCATCTATTGTTCGTTTTTCTATTTATC 
               
               
                   
               
               
                 TCACCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Somna 24R 
               
               
                 ATCCGTCACACCTGCTCTGGGTGAGATAAATAGAAAAACGAACAATA 
               
               
                   
               
               
                 GATGGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 25F 
               
               
                 ATACGGGAGCCAACACCACACATACGAGTTATCTCGAGTAGAGCATG 
               
               
                   
               
               
                 TTTTGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 25R 
               
               
                 ATCCGTCACACCTGCTCTGGCAAAACATGCTCTACTCGAGATAACTCG 
               
               
                   
               
               
                 TATGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 28F 
               
               
                 ATACGGGAGCCAACACCATCCATAGCTCATCTATACCCTCTTCCGAG 
               
               
                   
               
               
                 TCCCACCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 28R 
               
               
                 ATCCGTCACACCTGCTCTGGTGGGACTCGGAAGAGGGTATAGATGAG 
               
               
                   
               
               
                 CTATGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 33F 
               
               
                 ATACGGGAGCCAACACCAGAGCAGGTGTGACGGATAGTGACGGATGC 
               
               
                   
               
               
                 AGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 33R 
               
               
                 ATCCGTCACACCTGCTCTGCATCCGTCACTATCCGTCACACCTGCTCT 
               
               
                   
               
               
                 GGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 41F 
               
               
                 ATACGGGAGCCAACACCACCTTATGACGCCTCAGTACCACATCGTTT 
               
               
                   
               
               
                 AGTCTGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 41R 
               
               
                 ATCCGTCACACCTGCTCTACAGACTAAACGATGTGGTACTGAGGCGTC 
               
               
                   
               
               
                 ATAAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 45F 
               
               
                 ATACGGGAGCCAACACCACCCGTTTTTGATCTAATGAGGATACAATA 
               
               
                   
               
               
                 TTCGTCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 45R 
               
               
                 ATCCGTCACACCTGCTCTAGACGAATATTGTATCCTCATTAGATCAAA 
               
               
                   
               
               
                 AACGGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 46F 
               
               
                 ATACGGGAGCCAACACCATCGAGCTCCTTGGCCCCGTTAGGATTAAC 
               
               
                   
               
               
                 GTGATGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 46R 
               
               
                 ATCCGTCACACCTGCTCTACATCACGTTAATCCTAACGGGGCCAAGG 
               
               
                   
               
               
                 AGCTCGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 47F 
               
               
                 ATACGGGAGCCAACACCATCAGAACCAAATATACATCTTCCTATGAT 
               
               
                   
               
               
                 ATGGTGGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 47R 
               
               
                 ATCCGTCACACCTGCTCTCCACCATATCATAGGAAGATGTATATTTG 
               
               
                   
               
               
                 GTTCTGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 48F 
               
               
                 ATACGGGAGCCAACACCACACGATTGCTCCTCTCATTGTTACTTCATA 
               
               
                   
               
               
                 GCGACGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 48R 
               
               
                 ATCCGTCACACCTGCTCTCGTCGCTATGAAGTAACAATGAGAGGAGCA 
               
               
                   
               
               
                 ATCGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Teichoic Acid or Lipoteichoic Acid Binding 
               
               
                 Aptamer Sequences: 
               
               
                 T5 F 
               
               
                 GATACGGGACGACACCACACTATGGGTCGTTTAGCATCAAGGCTAGCC 
               
               
                   
               
               
                 AAGCCAGCAGAGGTGTGGTGAATG 
               
               
                   
               
               
                 T5 R 
               
               
                 CATTCACCACACCTCTGCTGGCTTGGCTAGCCTTGATGCTAAACGACC 
               
               
                   
               
               
                 CATAGTGTGGTGTCGTCCCGTATC 
               
               
                   
               
               
                 T6 F 
               
               
                 CATTCACCACACCTCTGCTGGAGGAGGAAGTGGTCTGGAGTTACTTGA 
               
               
                   
               
               
                 CATAGTGTGGTGTCGTCCCGTATC 
               
               
                   
               
               
                 T6 R 
               
               
                 GATACGGGACGACACCACACTATGTCAAGTAACTCCAGACCACTTCC 
               
               
                   
               
               
                 TCCTCCAGCAGAGGTGTGGTGAATG 
               
               
                   
               
               
                 T7 F 
               
               
                 CATTCACCACACCTCTGCTGGACGGAAACAATCCCCGGGTACGAGAAT 
               
               
                   
               
               
                 CAGGGTGTGGTGTCGTCCCGTATC 
               
               
                   
               
               
                 T7 R 
               
               
                 GATACGGGACGACACCACACCCTGATTCTCGTACCCGGGGATTGTTTC 
               
               
                   
               
               
                 CGTCCAGCAGAGGTGTGGTGAATG 
               
               
                   
               
               
                 T9 F 
               
               
                 CATTCACCACACCTCTGCTGGAAACCTACCATTAATGAGACATGATG 
               
               
                   
               
               
                 CGGTGGTGTGGTGTCGTCCCGTATC 
               
               
                   
               
               
                 T9 R 
               
               
                 GATACGGGACGACACCACACCACCGCATCATGTCTCATTAATGGTAG 
               
               
                   
               
               
                 GTTTCCAGCAGAGGTGTGGTGAATG 
               
               
                   
               
               
                   E. coli  O157 lipopolysaccharide (LPS) 
               
               
                 E-5F 
               
               
                 ATCCGTCACACCTGCTCTGGTGGAATGGACTAAGCTAGCTAGCGTTTT 
               
               
                   
               
               
                 AAAAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 E-11F 
               
               
                 ATCCGTCACACCTGCTCTGTAAGGGGGGGGAATCGCTTTCGTCTTAAG 
               
               
                   
               
               
                 ATGACATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 E-12F 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGTT 
               
               
                   
               
               
                 GGCTCCCGTAT(59) 
               
               
                   
               
               
                 E-16F 
               
               
                 ATCCGTCACACCTGCTCTATCCGTCACGCCTGCTCTATCCGTCACACC 
               
               
                   
               
               
                 TGCTCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 E-17F 
               
               
                 ATCCGTCACACCTGCTCTATCAAATGTGCAGATATCAAGACGATTTG 
               
               
                   
               
               
                 TACAAGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 E-18F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAAC 
               
               
                   
               
               
                 GATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 E-19F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAAC 
               
               
                   
               
               
                 GATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 E-5R 
               
               
                 ATACGGGAGCCAACACCACCTTTTAAAACGCTAGCTAGCTTAGTCCAT 
               
               
                   
               
               
                 TCCACCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 E-11R 
               
               
                 ATACGGGAGCCAACACCATGTCATCTTAAGACGAAAGCGATTCCCCC 
               
               
                   
               
               
                 CCCTTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 E-12R 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCA 
               
               
                   
               
               
                 GGTGTGACGGAT 
               
               
                   
               
               
                 E-16R 
               
               
                 ATACGGGAGCCAACACCAGAGCAGGTGTGACGGATAGAGCAGGCGTG 
               
               
                   
               
               
                 ACGGATAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 E-17R 
               
               
                 ATACGGGAGCCAACACCATCTTGTACAAATCGTCTTGATATCTGCAC 
               
               
                   
               
               
                 ATTTGATAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 E-18R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGC 
               
               
                   
               
               
                 CATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 E-19R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGC 
               
               
                   
               
               
                 CATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Listeriolysin (A surface protein on  Listeria   
               
               
                 
                   monocytogenes) 
                 
               
               
                 LO-10F 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTAT 
               
               
                   
               
               
                 LO-11F 
               
               
                 ATCCGTCACACCTGCTCTGGTGGAATGGACTAAGCTAGCTAGCGTTTT 
               
               
                   
               
               
                 AAAAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-13F 
               
               
                 ATCCGTCACACCTGCTCTTAAAGTAGAGGCTGTTCTCCAGACGTCGC 
               
               
                   
               
               
                 AGGAGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-15F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAAC 
               
               
                   
               
               
                 GATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-16F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACG 
               
               
                   
               
               
                 ATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-17F 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAG 
               
               
                   
               
               
                 GTGTGACGGAT 
               
               
                   
               
               
                 LO-19F 
               
               
                 ATCCGTCACACCTGCTCTTGGGCAGGAGCGAGAGACTCTAATGGTAAG 
               
               
                   
               
               
                 CAAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-20F 
               
               
                 ATCCGTCACACCTGCTCTCCAACAAGGCGACCGACCGCATGCAGATAG 
               
               
                   
               
               
                 CCAGGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-10R 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAG 
               
               
                   
               
               
                 GTGTGACGGAT 
               
               
                   
               
               
                 LO-11R 
               
               
                 ATACGGGAGCCAACACCACCTTTTAAAACGCTAGCTAGCTTAGTCCA 
               
               
                   
               
               
                 TTCCACCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-13R 
               
               
                 ATACGGGAGCCAACACCATCCTCCTGCGACGTCTGGAGAACAGCCTC 
               
               
                   
               
               
                 TACTTTAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-15R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCC 
               
               
                   
               
               
                 ATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-16R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTG 
               
               
                   
               
               
                 CCATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-17R 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTAT 
               
               
                   
               
               
                 LO-19R 
               
               
                 ATACGGGAGCCAACACCATTCTTGCTTACCATTAGAGTCTCTCGCT 
               
               
                   
               
               
                 CCTGCCCAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-20R 
               
               
                 ATACGGGAGCCAACACCAACCTGGCTATCTGCATGCGGTCGGTCGCC 
               
               
                   
               
               
                 TTGTTGGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Listeriolysin (Alternate form of  Listeria   
               
               
                 surface protein designated “Pest-Free”) 
               
               
                 LP-3F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACG 
               
               
                   
               
               
                 ATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LP-11F 
               
               
                 ATCCGTCACACCTGCTCTAACCAAAAGGGTAGGAGACCAAGCTAGCGA 
               
               
                   
               
               
                 TTTGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LP-13F 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGTT 
               
               
                   
               
               
                 GGCTCCCGTAT 
               
               
                   
               
               
                 LP-14F 
               
               
                 ATCCGTCACACCTGCTCTGAAGCCTAACGGAGAAGATGGCCCTACTGC 
               
               
                   
               
               
                 CGTAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LP-15F 
               
               
                 ATCCGTCACACCTGCTCTACTAAACAAGGGCAAACTGTAAACACAGT 
               
               
                   
               
               
                 AGGGGCGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LP-17F 
               
               
                 ATCCGTCACACCTGCTCTGGTGTTGGCTCCCGTATAGCTTGGCTCCCG 
               
               
                   
               
               
                 TATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LP-18F 
               
               
                 ATCCGTCACACCTGCTCTGTCGCGATGATGAGCAGCAGCGCAGGAGGG 
               
               
                   
               
               
                 AGGGGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LP-20F 
               
               
                 ATCCGTCACACCTGCTCTGATCAGGGAAGACGCCAACACTGGTGTTGG 
               
               
                   
               
               
                 CTCCCGTAT 
               
               
                   
               
               
                 LP-3R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCC 
               
               
                   
               
               
                 ATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LP-11R 
               
               
                 ATACGGGAGCCAACACCATCCAAATCGCTAGCTTGGTCTCCTACCCTT 
               
               
                   
               
               
                 TTGGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LP-13R 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAG 
               
               
                   
               
               
                 GTGTGACGGAT 
               
               
                   
               
               
                 LP-14R 
               
               
                 ATACGGGAGCCAACACCACCTACGGCAGTAGGGCCATCTTCTCCGTTA 
               
               
                   
               
               
                 GGCTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LP-15R 
               
               
                 ATACGGGAGCCAACACCACGCCCCTACTGTGTTTACAGTTTGCCCTTG 
               
               
                   
               
               
                 TTTAGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LP-17R 
               
               
                 ATACGGGAGCCAACACCATACGGGAGCCAAGCTATACGGGAGCCAACA 
               
               
                   
               
               
                 CCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LP-18R 
               
               
                 ATACGGGAGCCAACACCACCCCCTCCCTCCTGCGCTGCTGCTCATCAT 
               
               
                   
               
               
                 CGCGACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LP-20R 
               
               
                 ATACGGGAGCCAACACCAGTGTTGGCGTCTTCCCTGATCAGAGCAGG 
               
               
                   
               
               
                 TGTGACGGAT 
               
               
                   
               
               
                   Salmonella typhimurium  lipopolysaccharide (LPS) 
               
               
                 St-7F 
               
               
                 ATCCGTCACACCTGCTCTGTCCAAAGGCTACGCGTTAACGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTAT 
               
               
                   
               
               
                 St-10F 
               
               
                 ATCCGTCACACCTGCTCTGGAGCAATATGGTGGAGAAACGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTAT 
               
               
                   
               
               
                 St-11F 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGT 
               
               
                   
               
               
                 TGGCTCCCGTAT 
               
               
                   
               
               
                 St-15F 
               
               
                 ATCCGTCACACCTGCTCTGAACAGGATAGGGATTAGCGAGTCAACTAA 
               
               
                   
               
               
                 GCAGCATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-16F 
               
               
                 ATCCGTCACACCTGCTCTGGCGGACAGGAAATAAGAATGAACGCAAAA 
               
               
                   
               
               
                 TTTATCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-18F 
               
               
                 ATCCGTCACACCTGCTCTACGCAACGCGACAGGAACATTCATTATAGA 
               
               
                   
               
               
                 ATGTGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-19F 
               
               
                 ATCCGTCACACCTGCTCTCGGCTGCAATGCGGGAGAGTAGGGGGGAAC 
               
               
                   
               
               
                 CAAACCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-20F 
               
               
                 ATCCGTCACACCTGCTCTATGACTGGAACACGGGTATCGATGATTAGA 
               
               
                   
               
               
                 TGTCCTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-7R 
               
               
                 ATACGGGAGCCAACACCACGTTAACGCGTAGCCTTTGGACAGAGCAGG 
               
               
                   
               
               
                 TGTGACGGAT 
               
               
                   
               
               
                 St-10R 
               
               
                 ATACGGGAGCCAACACCACGTTTCTCCACCATATTGCTCCAGAGCAGG 
               
               
                   
               
               
                 TGTGACGGAT 
               
               
                   
               
               
                 St-11R 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAG 
               
               
                   
               
               
                 GTGTGACGGAT 
               
               
                   
               
               
                 St-15R 
               
               
                 ATACGGGAGCCAACACCATGCTGCTTAGTTGACTCGCTAATCCCTATC 
               
               
                   
               
               
                 CTGTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-16R 
               
               
                 ATACGGGAGCCAACACCAGATAAATTTTGCGTTCATTCTTATTTCCTG 
               
               
                   
               
               
                 TCCGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-18R 
               
               
                 ATACGGGAGCCAACACCAACACATTCTATAATGAATGTTCCTGTCGCG 
               
               
                   
               
               
                 TTGCGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-19R 
               
               
                 ATACGGGAGCCAACACCAGGTTTGGTTCCCCCCTACTCTCCCGCATTGC 
               
               
                   
               
               
                 AGCCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-20R 
               
               
                 ATACGGGAGCCAACACCAAGGACATCTAATCATCGATACCCGTGTTCC 
               
               
                   
               
               
                 AGTCATAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0014]      FIG. 1 . is a schematic illustration that illustrates a comparison of possible nucleic acid FRET assay formats. 
           [0015]      FIGS. 2A . and  2 B. are line graphs mapping relative fluorescence intensity against the concentration of surface protein from  L. donovani  from various freeze-dried and reconstituted competitive FRET-aptamer assays. 
           [0016]      FIGS. 3A . and  3 B. are “lights on” competitive FRET-aptamer spectra and a line graph for  E. coli  bacteria using aptamers generated against various components of lipopolysaccharide (LPS) such as the rough core (Ra) antigen and the 2-keto-3-deoxyoctanate (KDO) antigen. 
           [0017]      FIGS. 4A . and  4 B. are “lights on” competitive FRET-aptamer spectra and a bar graph for  Enterococcus faecalis  bacteria using aptamers generated against lipoteichoic acid. 
           [0018]      FIGS. 5A . and  5 B. are “lights off” competitive FRET-aptamer spectra and line graphs in response to increasing amounts of a foot-and-mouth disease (FMD) aphthovirus surface peptide. 
           [0019]      FIGS. 6A . and  6 B. are “lights on” competitive FRET-aptamer spectra and  FIG. 6C . is a line graph in response to increasing amounts of methylphosphonic acid (MPA; an organophosphorus (OP) nerve agent breakdown product). 
           [0020]      FIGS. 7A and 7B . are Sephadex G25 size-exclusion column profiles of complexes of Alexa Fluor (AF) 546-dUTP-labeled competitive FRET-aptamers bound to BHQ-2-amino-MPA (quencher-labeled target). The fractions with the highest absorbance at 260 nm (DNA aptamer), 555 nm (AF 546), and 579 nm (BHQ-2) were pooled and used in the competitive assay for unlabeled MPA, because these fractions contain the FRET-aptamer-quencher-labeled target complexes. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0021]    Referring to the figures,  FIG. 1 . provides a comparison of possible nucleic acid FRET assay formats. It illustrates how the competitive aptamer FRET scheme differs from other oligonucleotide-based FRET assay formats. Upper left is a molecular beacon ( 10 ) which may or may not be an aptamer, but is typically a short oligonucleotide used to hybridize to other DNA or RNA molecules and exhibit FRET upon hybridizing. Molecular beacons are only labeled with F and Q at the ends of the DNA molecule. Lower left is a signaling aptamer ( 12 ), which does not contain a quencher molecule, but relies upon fluorophore self-quenching or weak intrinsic quenching of the DNA or RNA to achieve limited FRET. Upper right is an intrachain FRET-aptamer ( 14 ) containing F and Q molecules built into the interior structure of the aptamer. Intrachain FRET-aptamers are naturally selected and characterized by the processes described herein. Lower right shows a competitive aptamer FRET ( 16 ) motif in which the aptamer container either F or Q and the target molecule ( 18 ) is labeled with the complementary F or Q. Introduction of unlabeled target molecules ( 20 ) then shifts the equilibrium so that some labeled target molecules are liberated from the labeled aptamer and modulate the fluorescence level of the solution up or down thereby achieving FRET. A target analyte ( 20 ) is either unlabeled or labeled with a quencher (Q). F and Q can be switched from placement in the aptamer to placement in the target analyte and vice versa. 
         [0022]    F-labeled or Q-labeled aptamers (labeled by the polymerase chain reaction (PCR), asymmetric PCR (to produce a predominately single-stranded amplicon) using Taq, Deep Vent Exo −  or other heat-resistant DNA polymerases, or other enzymatic incorporation of F-NTPs or Q-NTPs) may be used in competitive or displacement type assays in which the fluorescence light levels change proportionately in response to the addition of various levels of unlabeled analyte which compete to bind with the F-labeled or Q-labeled analytes. 
         [0023]    Competitive aptamer-FRET assays may be used for the detection and quantitation of small molecules (&lt;1,000 daltons) including pesticides, acetylcholine (ACh), organophosphate (“OP”) nerve agents such as sarin, soman, and VX, OP nerve agent breakdown products such as MPA, isopropyl-MPA, ethylmethyl-MPA, pinacolyl-MPA, etc., acetylcholine (ACh), acyl homoserine lactone (AHL) and other quorum sensing (QS) molecules natural and synthetic amino acids and their derivatives (e.g., histidine, histamine, homocysteine, DOPA, melatonin, nitrotyrosine, etc.), short chain proteolysis products such as cadaverine, putrescine, the polyamines spermine and spermidine, nitrogen bases of DNA or RNA, nucleosides, nucleotides, and their cyclical isoforms (e.g., cAMP and cGMP), cellular metabolites (e.g., urea, uric acid), pharmaceuticals (therapeutic drugs), drugs of abuse (e.g., narcotics, hallucinogens, gamma-hydroxybutyrate, etc.), cellular mediators (e.g., cytokines, chemokines, immune modulators, neural modulators, inflammatory modulators such as prostaglandins, etc.), or their metabolites, explosives (e.g., trinitrotoluene) and their breakdown products or byproducts, peptides and their derivatives, macromolecules including proteins (such as bacterial surface proteins from  Leishmania donovani,  See  FIGS. 2A and 2B ), glycoproteins, lipids, glycolipids, nucleic acids, polysaccharides, lipopolysaccharides (LPS), and LPS components (e.g., ethanolamine, glucosamine, LPS-specific sugars, KDO, rough core antigens, etc.), viruses, whole cells (bacteria and eukaryotic cells, cancer cells, etc.), and subcellular organelles or cellular fractions. 
         [0024]    If the target molecule is a larger water-soluble molecule such as a protein, glycoprotein, or other water soluble macromolecule, then exposure of the nascent F-labeled and Q-labeled DNA or RNA random library to the free target analyte is done in solution. If the target is a soluble protein or other larger water-soluble molecule, then the optimal FRET-aptamer-target complexes are separated by size-exclusion chromatography. The FRET-aptamer-target complex population of molecules is the heaviest subset in solution and will emerge from a size-exclusion column first, followed by unbound FRET-aptamers and unbound proteins or other targets. Among the subset of analyte-bound aptamers there will be heterogeneity in the numbers of F- and Q-NTP&#39;s that are incorporated as well as nucleotide sequence differences, which will again effect the mass, electrical charge, and weak interaction capabilities (e.g., hydrophobicity and hydrophilicity) of each analyte-aptamer complex. These differences in physical properties of the aptamer-analyte complexes can then be used to separate out or partition the bound from unbound analyte-aptamer complexes. 
         [0025]    If the target is a small molecule, then exposure of the nascent F-labeled and Q-labeled DNA or RNA random library to the target may be done by immobilizing the target. The small molecule can be immobilized on a column, membrane, plastic or glass bead, magnetic bead, quantum dot, or other matrix. If no functional group is available on the small molecule for immobilization, the target can be immobilized by the Mannich reaction (formaldehyde-based condensation reaction) on a PharmaLink™ column. Elution of bound DNA from the small molecule affinity column, membrane, beads or other matrix by use of 0.2-3.0M sodium acetate at a pH of between 3 and 7. 
         [0026]    These can be separated from the non-binding doped DNA molecules by running the aptamer-protein aggregates (or selected aptamers-protein aggregates) through a size exclusion column, by means of size-exclusion chromatography using Sephadex™ or other gel materials in the column. Since they vary in weight due to variations in aptamers sequences and degree of labeling, they can be separated into fractions with different fluorescence intensities. Purification methods such as preparative gel electrophoresis are possible as well. Small volume fractions (≦1 mL) can be collected from the column and analyzed for absorbance at 260 nm and 280 nm which are characteristic wavelengths for DNA and proteins. In addition, the characteristic absorbance wavelengths for the fluorophore and quencher ( FIGS. 7A and 7B ) should be monitored. The heaviest materials come through a size-exclusion column first. Therefore, the DNA-protein complexes will come out of the column before either the DNA or protein alone. 
         [0027]    Means of separating FRET-aptamer-target complexes from solution by alternate techniques (other than size-exclusion chromatography) include, without limitation, molecular weight cut off spin columns, dialysis, analytical and preparative gel electrophoresis, various types of high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and differential centrifugation using density gradient materials. 
         [0028]    The optimal (most sensitive or highest signal to noise ratio) FRET-aptamers among the bound class of FRET-aptamer-target complexes are identified by assessment of fluorescence intensity for various fractions of the FRET-aptamer-target class. The separated DNA-protein complexes will exhibit the highest absorbance at established wavelengths, such as 260 nm and 280 nm. The fractions showing the highest absorbance at the given wavelengths, such as 260 nm and 280 nm, are then further analyzed for fluorescence and those fractions exhibiting the greatest fluorescence are selected for separation and sequencing. 
         [0029]    These similar FRET-aptamers may be further separated using techniques such as ion pair reverse-phase high performance liquid chromatography, ion-exchange chromatography (IEC, either low pressure or HPLC versions of IEC), thin layer chromatography (TLC), capillary electrophoresis, or similar techniques. 
         [0030]    The final FRET aptamers are able to act as one-step “lights on” or “lights off” binding and detection components in assays. 
         [0031]    Intrachain FRET-aptamers that are to be used in assays with long shelf-lives may be lyophilized (freeze-dried) and reconstituted. 
         [0032]      FIGS. 2A . and  2 B. are line graphs mapping the fluorescence intensity of the DNA aptamers against the concentration of the surface protein. The figures present results from two independent trials of a competitive aptamer-FRET assay involving fluorophore-labeled DNA aptamers and surface extracted proteins from  Leishmania donovani  bacteria. In this type of assay, the fluorescence intensity decreases as a function of increasing analyte concentration, and is thus referred to as a “lights off” assay. If the fluorescence intensity increases as a function of increasing analyte concentration, then it is referred to as a “lights on” assay. Also shown are translations of the assay curve up or down due to lyophilization (freeze-drying) in the absence or presence of 10% fetal bovine serum (FBS). Error bars represent the standard deviations of the mean for three measurements. 
         [0033]      FIGS. 3A . and  3 B. are FRET fluorescence spectra and line graphs generated as a function of live  E. coli  (Crooks strain, ATCC No. 8739) concentration using LPS component competitive FRET-aptamers. Error bars represent the standard deviations of the mean for four measurements. 
         [0034]      FIGS. 4A . and  4 B. are FRET fluorescence spectra and line graphs generated as a function of live  Enterococcus faecalis  concentration using lipoteichoic acid (TA) competitive FRET-aptamers. Error bars represent the standard deviations of the mean for four measurements. 
         [0035]      FIGS. 5A . and  5 B. are FRET fluorescence spectra and line graphs generated as a function of Foot-and-Mouth Disease (FMD) peptide concentration using FMD peptide competitive FRET-aptamers. Error bars represent the standard deviations of the mean for four measurements. 
         [0036]      FIGS. 6A . and  6 B. are FRET fluorescence spectra, and  FIG. 6C . is a line graph, all generated as a function of methylphosphonic acid (MPA; OP nerve agent degradation product) concentration using MPA competitive FRET-aptamers to represent possible FRET-aptamer assays for MPA and OP nerve agents such as pesticides, sarin, soman, VX, etc. Error bars represent the standard deviations of the mean for four measurements. 
         [0037]      FIGS. 7A . and  7 B. are two independent Sephadex™ G25 elution profiles for BHQ-2-amino-MPA—AF 546-MPA aptamer complex based on absorbance peaks characteristic of the aptamer (260 nm), fluorophore (555 nm), and quencher (579 nm) to assess the optimal fraction for competitive FRET-aptamer assay of MPA shown in  FIG. 6 . Similar elution profiles can be expected for all such soluble targets when the target is quencher-labeled and complexed to a fluorophore-labeled aptamer. 
       EXAMPLE 1  
       [0038]    Competitive Aptamer-FRET Assay for Surface Proteins Extracted from Bacteria ( L. Donovani ). 
         [0039]    In this example, surface proteins from heat-killed  Leishmania donovani  were extracted with 3 M MgCl 2  overnight at 4° C. These proteins were then linked to tosyl-magnetic microbeads and used in a standard SELEX aptamer generation protocol. After 5 rounds of SELEX, the aptamer population was “doped” during the standard PCR reaction with 3 uM fluorescein-dUTP and purified on 10 kD molecular weight cut off spin columns. Some of the  L. donovani  surface proteins were then labeled with dabcyl-NHS ester and purified on a PD-10 (Sephadex G25) column. The dabcyl-labeled surface proteins were combined with the fluorescein-labeled aptamer population so as to produce a 1:1 fluorescein-aptamer:dabcyl-protein ratio. Thereafter, unlabeled  L. donovani  surface proteins were introduced into the assay system to compete with the labeled proteins for binding to the aptamers, thereby producing the “lights off” FRET assay results depicted in  FIGS. 2A and 2B  (fresh assay results, solid line). The assays were also examined following lyophilization (freeze drying) and reconstitution (rehydration) in the presence or absence of 10% fetal bovine serum (FBS) as a possible preservative with the results shown in  FIGS. 2A and 2B . The DNA sequences of several of these candidate  Leishmania  aptamers are given in SEQ IDs XX-XX. 
       EXAMPLE 2 
       [0040]    Competitive FRET-Aptamer Assay for  E. Coli  in Environmental Water Samples or Foods Using LPS Component Aptamers. 
         [0041]      E. coli,  especially the enterohemorrhagic strains such as O157:H7 which produce Verotoxin or Shiga toxins, are of concern in environmental water samples and foods. Their rapid detection (within minutes) with ultrasensitivity is important in protecting swimmers as well as those consuming water and foods. In this example, aptamers were generated against whole LPS from  E. coli  O111:B4 and its components such as glucosamine, KDO, and the rough mutant core antigen (Ra; lacking the outer oligosaccharide chains). In the case of glucosamine, the free primary amine in its structure enabled conjugation to tosyl-magnetic beads. KDO antigen was immobilized onto amine-conjugated magnetic beads via its carboxyl group and the bifunctional linker EDC. The rough Ra core antigen and whole LPS were linked to amine-magnetic beads via reductive amination using sodium periodate to oxidize the saccharides to aldehydes followed by the use of sodium cyanoborohydride for reductive amination as will be clear to anyone skilled in the art. Once immobilized the target-magnetic beads were used for aptamer affinity selection from a random library of 72 base aptamers (randomized 36mer flanked by known 18mer primer regions). After 5 rounds of aptamer selection and amplification, the various LPS component aptamer populations were subjected to 10 rounds of PCR in the presence of Alexa. Fluor (AF) 546-14-dUTP (Invitrogen), then heated to 95° C. for 5 minutes and added to heat-killed  E. coli  O157:H7 (Kirkegaard Perry Laboraties, Inc., Gaithersburg, Md.) and used in competitive FRET-aptamer assays with various concentrations of unlabeled live  E. coli  (Crooks strain, ATCC No. 8739) resulting in the FRET spectra and line graphs shown in  FIGS. 3A and 3B . Candidate DNA aptamer sequences for detection of LPS O111 and LPS components or associated  E. coli  and other Gram negative bacteria are given in SEQ ID Nos. XX-XX. 
       EXAMPLE 3 
       [0042]    Competitive FRET-Aptamer Assay for Enterococci in Environmental Water Samples 
         [0043]    Gram positive enterococci, such as  Enterococcus faecalis,  are also indicators of fecal contamination of environmental water, recreational waters, or treated wastewater (effluent from sewage treatment plants). Water testers desire to detect the presence of these bacteria rapidly (within minutes) and with great sensitivity. In this example, aptamers were generated against whole lipoteichoic acid (TA; teichoic acid). TA from  E. faecalis  was immobilized on magnetic beads by reductive amination using sodium periodate to first oxidize saccharides into aldehydes followed by reductive amination using amine-magnetic beads and sodium cyanoborohydride as will be known to anyone skilled in the art. Once immobilized the target-magnetic beads were used for aptamer affinity selection from a random library of 72 base aptamers (randomized 36mer flanked by known 18mer primer regions). After 5 rounds of aptamer selection and amplification, the TA aptamer population was subjected to 10 rounds of PCR in the presence of AF 546-14-dUTP (Invitrogen), then heated to 95° C. for 5 minutes and added to live  E. faecalis . The complexes were purified by centrifugation and washing and used in competitive FRET-aptamer assays with various concentrations of unlabeled live  E. faecalis  resulting in the FRET spectra and bar graphs shown in  FIGS. 4A . and  4 B. Candidate DNA aptamer sequences for detection of lipoteichoic acid (TA) and associated  enterococi  or other Gram positive bacteria are given in SEQ ID Nos. XX-XX. 
       EXAMPLE 4 
       [0044]    Detection of Foot-and-Mouth (FMD) Disease or Other Highly Communicable Viruses Among Animal or Human Populations 
         [0045]    FMD has not existed in the United States for decades, but if it were reintroduced via agricultural bioterrorism or accidental means, it could cripple the multi-billion dollar livestock industry. Hence, rapid detection of FMD in the field (on farms) is of great value in quarantining infected animals or farms and limiting the spread of FMD. Likewise, epidemiologists have many uses for rapid field detection and identification of viruses and other microbes such as influenzas, potential small pox outbreaks, etc. which FRET-aptamer assays could satisfy. A highly conserved peptide from the VP1 structural protein of O-type FMD, which is widely distributed throughout the world, was chosen as the aptamer development target. The peptide had the following primary amino acid sequence: RHKQKIVAPVKQLL. This sequence corresponds to amino acids 200 through 213 of 16 different O-type FMD viruses and represents a neutralizable antigenic region wherein antibodies are known to bind. The FMD peptide was immobilized on tosyl-magnetic beads via the three lysine residues in its structure. Once immobilized the target-magnetic beads were used for aptamer affinity selection from a random library of 72 base aptamers (randomized 36mer flanked by known 18mer primer regions). After 5 rounds of aptamer selection and amplification, the FMD (peptide) aptamer populations were subjected to 10 rounds of PCR in the presence of Alexa Fluor (AF) 546-14-dUTP (Invitrogen), then heated to 95° C. for 5 minutes and added to their BHQ-2-labeled-peptide target. The complexes were purified by size-exclusion chromatography over Sephadex G25 and used in competitive FRET-aptamer assays with various concentrations of unlabeled FMD peptide resulting in the FRET spectra and line graphs shown in  FIGS. 5A and 5B . Candidate DNA aptamer sequences for detection of the FMD peptide and associated strains of FMD virus are given in SEQ ID Nos. XX-XX. 
       EXAMPLE 5 
       [0046]    Detection of Organophosphorus (OP) Nerve Agent, Pesticides, and Acetylcholine (ACh) 
         [0047]    The use of OP nerve agents on Iraqi Kurds in the late 1980&#39;s followed by the 1995 use of sarin in a Japanese subway underscore the need for rapid and sensitive detection of OP nerve agents such as FRET-aptamer assays might provide. In addition, there is a desire in the agricultural industry to detect pesticides (also OP nerve agents) on the surfaces of fruits and vegetables in the field or in grocery stores. Finally, aptamers that bind and detect acetylcholine (ACh) may be of value in determining the impact of OP nerve agents on acetylcholinesterase (AChE) activity. Candidate aptamer sequences for the nerve agent soman, methylphosphonic acid (MPA, a common nerve agent hydrolysis product), the pesticides diazinon and malathion, and ACh are given in SEQ ID Nos. XX-XX. Amino-MPA and para-aminophenyl-soman were immobilized on tosyl-magnetic beads and used for aptamer selelction. ACh and the pesticides were immobilized onto PharmaLink™ (Pierce Chemical Co.) affinity columns by the Mannich formaldehyde condensation reaction and used for aptamer selection. The polyclonal or monoclonal candidate MPA aptamers were labeled with AF 546-14-dUTP by 10 rounds of conventional PCR or 20 rounds of asymmetric as appropriate with Deep Vent Exo −  polymerase and then complexed to BHQ-2-amino-MPA. The complexes were purified by size-exclusion chromatography over Sephadex G-15 and used to generate FRET spectra and line graphs as a function of unlabeled MPA as shown in FIGS.  6 A.,  6 B., and  6 C. 
         [0048]    Other potential examples of uses for competitive FRET-aptamer assays include, but are not limited to:
   1) Detection and quantitation of quorum sensing (QS) molecules such as acyl homoserine lactones (AHLs such as N-Decanoyl-DL-Homoserine Lactone; SEQ ID Nos. XX-XX), which communicate between many Gram negative bacteria such as Pseudomonads to signal proliferation and the induction of virulence factors, thereby leading to disease.   2) Detection and quantitation of botulinum toxins (BoNTs) for determination of the presence of biological warfare or bioterrorism agents (SEQ ID Nos. XX-XX) and  Clostridium botulinum  in vivo.   3) Detection and quantitation of  Campylobacter jejuni  and related  Campylobacter  species (SEQ ID Nos. XX-XX) in foods and water to prevent foodborne or waterborne illness outbreaks (add 2006 JCLA paper reference here).   4) Detection and quantitation of poly-D-glutamic acid (PDGA; SEQ ID Nos. XX-XX) from vegetative forms of pathogenic  Bacillus anthracis  or other similar encapsulated bacteria in vivo or in the environment to rapidly diagnose biological warfare or bioterrorist activity and provide intervention.   5) Detection and quantitation of  Bacillus thuringiensis  bacterial endospores in the environment to assist in biological warfare or bioterrorism detection field trials or forensic work.   
 
         [0054]    Although the invention has been described with reference to specific embodiments, this description is 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 the 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.