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 Exo −  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 co-pending U.S. application Ser. No. 13/373,993, filed on Dec. 7, 2011, which is a continuation of now-abandoned U.S. application Ser. No. 12/011,675, filed on Jan. 29, 2008, which is a continuation-in-part of co-pending U.S. application Ser. No. 11/433,283, filed on May 12, 2006. Each patent application identified above is incorporated here by reference in its entirety to provide continuity of disclosure. 
     
    
     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). A fluorophore is a molecule (e.g., colored dye) which emits light at a specific range of wavelengths or segment of the spectrum after excitation by light of a lower wavelength or lower range of wavelengths versus the emission wavelengths. Different types of fluorophores emit energy at different wavelengths or spectral ranges. A quencher is a molecule which absorbs light energy (or photons) at a specific spectral range of wavelengths and does not re-emit light, but converts virtually all of the excitation light energy into invisible vibrations (e.g., infrared or heat). Different types of quenchers absorb energy at different wavelengths or spectral ranges. 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). In order to achieve FRET, 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 (greater than 50%) with the absorption spectrum of Q, such that when the F and the Q are moved into or out of functional proximity (the Förster distance of less than or equal to 85 Angstroms), there is a detectable change in the fluorescent signal of the aptamer—either more detectable light when the Q is moved away from the F, or less detectable light when the Q is moved near the F. 
         [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&#39;s, Cascade Blue®-NTP&#39;s, 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. 
         [0014]    The invention described herein may use one or more aptamer sequences. The aptamers are identified in the accompanying Sequence Listing. The aptamers in the Sequence Listing are listed 5′ to 3′ from left to right. Aptamers that target Acetylcholine (ACh) are identified in the Sequence Listing as SEQ ID NO&#39;s 1 to 26. Aptamers that target Acyl Homoserine Lactone (AHL) Quorum Sensing Molecules (N-Decanoyl-DL-Homoserine Lactone) are identified in the Sequence Listing as SEQ ID NO&#39;s 27 to 36. An aptamer that targets  Bacillus thuringiensis  spores is identified in the Sequence Listing as SEQ ID NO 37. An aptamer that targets Botulinum Toxin (BoNT Type A) is identified in the Sequence Listing as SEQ ID NO 38. Aptamers that target Botulinum Toxin are identified in the Sequence Listing as SEQ ID NO&#39;s 38 to 41. Aptamers that target  Campylobacter jejuni , are identified in the Sequence Listing as SEQ ID NO&#39;s 42 to 47. Aptamers that target Diazinon (D) are identified in the Sequence Listing as SEQ ID NO&#39;s 48 to 59. Aptamers that target Glucosamine, are identified in the Sequence Listing as SEQ ID NO&#39;s 60 to 75. Aptamers that target KDO Antigen are identified in the Sequence Listing as SEQ ID NO&#39;s 76 to 87. Aptamers that target  Leishmania donovani  are identified in the Sequence Listing as SEQ ID NO&#39;s 88 to 91. Aptamers that target lipopolysaccharide (LPS) from  E. coli  0111 are identified in the Sequence Listing as SEQ ID NO&#39;s 92 to 107. Aptamers that target Methylphosphonic Acid (MPA) are identified in the Sequence Listing as SEQ ID NO&#39;s 108 to 109. Aptamers that target Malathion are identified in the Sequence Listing as SEQ ID NO&#39;s 110 to 115. Aptamers that target Poly-D-Glutamic Acid are identified in the Sequence Listing as SEQ ID NO&#39;s 116 to 119. Aptamers that target Rough Ra Mutant LPS Core are identified in the Sequence Listing as SEQ ID NO&#39;s 120 to 133. Aptamers that target Soman are identified in the Sequence Listing as SEQ ID NO&#39;s 134 to 155. Aptamers that target Teichoic Acid or Lipoteichoic Acid are identified in the Sequence Listing as SEQ ID NO&#39;s 156 to 163. Aptamers that target  E. coli  O157 lipopolysaccharide are identified in the Sequence Listing as SEQ ID NO&#39;s 164 to 177. Aptamers that target Listeriolysin (a surface protein on  Listeria monocytogenes ) are identified in the Sequence Listing as SEQ ID NO&#39;s 178 to 193. Aptamers that target Listeriolysin (an alternate form of  Listeria  surface protein designated “Pest-Free”) are identified in the Sequence Listing as SEQ ID NO&#39;s 194 to 209. Aptamers that target  Salmonella typhimurium  lipopolysaccharide are identified in the Sequence Listing as SEQ ID NO&#39;s 210 to 225. 
       Acetylcholine (ACh) Aptamer Sequences: 
       [0015]      
         [0000]    
       
         
               
               
             
           
               
                   
                 ACh1a For 
               
               
                   
                 ATACGGGAGCCAACACCACGATACCCGCTTATGAATTTTAAATTA 
               
               
                   
                 ATTGTGATCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh1a Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTGATCACAATTAATTTAAAATTCATAAG 
               
               
                   
                 CGGGTATCGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 1b For 
               
               
                   
                 ATACGGGAGCCAACACCAACTTTCACACATACTTGTTATACCACA 
               
               
                   
                 CGATCTTTTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 1b Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTAAAAGATCGTGTGGTATAACAAGTATG 
               
               
                   
                 TGTGAAAGTTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 2 For 
               
               
                   
                 ATACGGGAGCCAACACCACTTTGTAACTCATTTGTAGTTTGGGTT 
               
               
                   
                 GCTCCCCCTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 2 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTAGGGGGAGCAACCCAAACTACAAATGA 
               
               
                   
                 GTTACAAAGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 3 For 
               
               
                   
                 ATACGGGAGCCAACACCATTTCCCGCTTATCTTCATCCACTGCTT 
               
               
                   
                 AGCATATGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 3 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTACATATGCTAAGCAGTGGATGAAGATA 
               
               
                   
                 AGCGGGAAATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 5 For 
               
               
                   
                 ATACGGGAGCCAACACCAGGCACTGTATCACACCGTCAAGAATGT 
               
               
                   
                 GATCCCCTGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 5 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTCAGGGGATCACATTCTTGACGGTGTGA 
               
               
                   
                 TACAGTGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 6 For 
               
               
                   
                 ATACGGGAGCCAACACCATGTCATTTACCTTCATCATGACAGTGT 
               
               
                   
                 TAGTATACGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 6Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTAGGGGATCAAAGCTATGCGACCATGCG 
               
               
                   
                 AGTGGATACTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 7 For 
               
               
                   
                 ATACGGGAGCCAACACCAGTTGCCGCCTACCTTGATTATTCTACA 
               
               
                   
                 TTACCCGTTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 7 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTAACGGGTAATGTAGAATAATCAAGGTA 
               
               
                   
                 GGCGGCAACTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 8 For 
               
               
                   
                 ATACGGGAGCCAACACCAGTATACATACGAAGAGTTGAAACCAAT 
               
               
                   
                 GCTTCGTTCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 8 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTGAACGAAGCATTGGTTTCAACTCTTCG 
               
               
                   
                 TATGTATACTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 9 For 
               
               
                   
                 ATACGGGAGCCAACACCATACCCCGAATGGCTGTTTTCAGTACCA 
               
               
                   
                 ATATGACTCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 9 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTGAGTCATATTGGTACTGAAAACAGCCA 
               
               
                   
                 TTCGGGGTATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 10 For 
               
               
                   
                 ATACGGGAGCCAACACCACTGTCACGATCGTCGTTCCTTTTAATC 
               
               
                   
                 CTTGTGTCTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 10 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTAGACACAAGGATTAAAAGGAACGACGA 
               
               
                   
                 TCGTGACAGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 11 For 
               
               
                   
                 ATACGGGAGCCAACACCACTGGACACTGACCCTCGCACTAGCTTT 
               
               
                   
                 CTGACGGGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 11 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTACCCGGCCGAAGAATAGTGCTCGGTAC 
               
               
                   
                 TTAGTCGCGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 ACh 12 For 
               
               
                   
                 ATACGGGAGCCAACACCATTTGGACTTTAAATAGTGGACTCCTTC 
               
               
                   
                 TTTGTCTCGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 ACh 12 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTCGAGACAAAGAAGGAGTCCACTATTTA 
               
               
                   
                 AAGTCCAAATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 A25 For 
               
               
                   
                 ATACGGGAGCCAACACCA-TCATTTGCAAATATGAATTCCACTTA 
               
               
                   
                 AAGAAATTCA-AGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 A25 Rev 
               
               
                   
                 ATCCGTCACACCTGCTCTTGAATTTCTTTAAGTGGAATTCATATT 
               
               
                   
                 TGCAAATGATGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Acyl Homoserine Lactone (AHL) Quorum Sensing Molecules (N-Decanoyl-DL-Homoserine Lactone) 
       [0016]      
         [0000]                    Dec AHL 1For       ATACGGGAGCCAACACCATCCTAACTGGTCTAATTTTTG       CTGTTACCGATCCCGAGAGCAGGTGTGACGGAT               Dec AHL 1 Rev       ATCCGTCACTCCTGCTCTCGGGATCGGTAACAGCAAAAA       TTAGACCAGTTAGGATGGTGTTGGCTCCCGTAT               Dec AHL 13 For       ATACGGGAGCCAACACCAGCCTGACGAAAAAATTTTATC       ACTAAGTGATACGCAAGAGCAGGTGTGACGGAT               Dec AHL 13 Rev       ATCCGTCACACCTGCTCTTGCGTATCACTTAGTGATAAA       ATTTTTTCGTCAGGCTGGTGTTGGCTCCCGTAT               Dec AHL 14 For       ATACGGGAGCCAACACCAGACCTACTTCAGAAACGGAAA       TGTTCTTAGCCGTCAGAGCAGGTGTGACGGAT               Dec AHL 14 Rev       ATCCGTCACACCTGCTCTGACGGCTAAGAACATTTCCGT       TTCTGAAGTAGGTCTGGTGTTGGCTCCCGTAT               Dec AHL 15 For       ATACGGGAGCCAACACCAGGCCAACGAAACTCCTACTAC       ATATAATGCTTATGCAGAGCAGGTGTGACGGAT               Dec AHL 15 Rev       ATCCGTCACACCTGCTCTGCATAAGCATTATATGTAGTA       GGAGTTTCGTTGGCCTGGTGTTGGCTCCCGTAT               Dec AHL 17 For       ATACGGGAGCCAACACCATCCTAACTGGTCTAATTTTTG       CTGTTACCGATCCCGAGAGCAGGTGTGACGGAT               Dec AHL 17 Rev       ATCCGTCACACCTGCTCTCGGGATCGGTAACAGCAAAAA       TTAGACCAGTTAGGATGGTGTTGGCTCCCGTAT              Bacillus thuringiensis  Spore Aptamer Sequence:
 
         [0000]    
       
         
               
               
             
           
               
                   
                 CATCCGTCACACCTGCTCTGGCCACTAACATGGGGACCAGGTGGT 
               
               
                   
                 GTTGGCTCCCGTATC 
               
             
          
         
       
     
       Botulinum Toxin (BoNT Type A) Aptamer Sequences: 
       [0017]    BoNT A Holotoxin (Heavy Chain plus Light Chain Linked Together) 
         [0000]    
       
         
               
               
             
           
               
                   
                 CATCCGTCACACCTGCTCTGCTATCACATGCCTGCTGAAGTGGTG 
               
               
                   
                 TTGGCTCCCGTATCA 
               
             
          
         
       
     
       BoNT A 50 kd Enzymatic Light Chain 
       [0018]      
         [0000]                            BoNT A Light Chain 1           CATCCGTCACACCTGCTCTGGGGATGTGTGGTGTTGGCTCCCGTA           TCAAGGGCGAATTCT                       BoNT A Light Chain 2           CATCCGTCACACCTGCTCTGATCAGGGAAGACGCCAACACGTGGT           GTTGGCTCCCGTATCA                       BoNT A Light Chain 3           CATCCGTCACACCTGCTCTGGGTGGTGTTGGCTCCCGTATCAAGG           GCGAATTCTGCAGATA              Campylobacter jejuni  Binding Aptamers:
 
         [0000]    
       
         
               
               
             
           
               
                   
                 C1 
               
               
                   
                 CATCCGTCACACCTGCTCTGGGGAGGGTGGCGCCCGTCTCGGTGG 
               
               
                   
                 TGTTGGCTCCCGTATCA 
               
               
                   
                   
               
               
                   
                 C 2 
               
               
                   
                 CATCCGTCACACCTGCTCTGGGATAGGGTCTCGTGCTAGATGTGG 
               
               
                   
                 TGTTGGCTCCCGTATCA 
               
               
                   
                   
               
               
                   
                 C 3 
               
               
                   
                 CATCCGTCACACCTGCTCTGGACCGGCGCTTATTCCTGCTTGTGG 
               
               
                   
                 TGTTGGCTCCCGTATCA 
               
               
                   
                   
               
               
                   
                 C 4 
               
               
                   
                 CATCCGTCACACCTGCYCTGGAGCTGATATTGGATGGTCCGGTGG 
               
               
                   
                 TGTTGGCTCCCGTATCA 
               
               
                   
                   
               
               
                   
                 C 5 
               
               
                   
                 CATCCGTCACACCTGCYCYGCCCAGAGCAGGTGTGACGGATGTGG 
               
               
                   
                 TGTTGGCTCCCGTATCA 
               
               
                   
                   
               
               
                   
                 C 6 
               
               
                   
                 CATCCGTCACACCTGCYCYGCCGGACCATCCAATATCAGCTGTGG 
               
               
                   
                 TGTTGGCTCCCGTATCA 
               
             
          
         
       
     
       Diazinon Binding Aptamers 
       [0019]      
         [0000]                            D12 Forward           ATACGGGAGCCAACACCATTAAATCAATTGTGCCGTGTTGGTCTT           GTCTCATCGAGAGCAGGTGTGACGGAT                       D12 Reverse           ATCCGTCACACCTGCTCTCGATGAGACAAGACCAACACGGCACAA           TTGATTTAATGGTGTTGGCTCCCGTAT                       D17 Forward           ATACGGGAGCCAACACCATTTTTATTATCGGTATGATCCTACGAG           TTCCTCCCAAGAGCAGGTGTGACGGAT                       D17 Reverse           ATCCGTCACACCTGCTCTTGGGAGGAACTCGTAGGATCATACCGA           TAATAAAAATGGTGTTGGCTCCCGTAT                       D18 Forward           ATACGGGAGCCAACACCACCGTATATCTTATTATGCACAGCATCA           CGAAAGTGCAGAGCAGGTGTGACGGAT                       D18 Reverse           ATCCGTCACACCTGCTCTGCACTTTCGTGATGCTGTGCATAATAA           GATATACGGTGGTGTTGGCTCCCGTAT                       D19 Forward           ATACGGGAGCCAACACCATTAACGTTAAGCGGCCTCACTTCTTTT           AATCCTTTCAGAGCAGGTGTGACGGAT                       D19 Reverse           ATCCGTCACACCTGCTCTGAAAGGATTAAAAGAAGTGAGGCCGCT           TAACGTTAATGGTGTTGGCTCCCGTAT                       D20 Forward           ATCCGTCACACCTGCTCTAATATAGAGGTATTGCTCTTGGACAAG           GTACAGGGATGGTGTTGGCTCCCGTAT                       D20 Reverse           ATACGGGAGCCAACACCATCCCTGTACCTTGTCCAAGAGCAATAC           CTCTATATTAGAGCAGGTGTGACGGAT                       D25 Forward           ATACGGGAGCCAACACCATTAACGTTAAGCGGCCTCACTTCTTTT           AATCCTTTCAGAGCAGGTGTGACGGAT                       D25 Reverse           ATCCGTCACACCTGCTCTGAAAGGATTAAAAGAAGTGAGGCCGCT           TAACGTTAATGGTGTTGGCTCCCGTAT            
Glucosamine (from LPS) Forward Aptamer Sequences:
 
         [0000]                            G 1 For           ATCCGTCACACCTGCTCTAATTAGGATACGGGGCAACAGAACGAG           AGGGGGGAATGGTGTTGGCTCCCGTAT                       G 2 For           ATCCGTCACACCTGCTCTCGGACCAGGTCAGACAAGCACATCGGA           TATCCGGCTGGTGTTGGCTCCCGTAT                       G 4 For           ATCCGTCACACCTGCTCTAATTAGGATACGGGGCAACAGAACGAG           AGGGGGGAATGGTGTTGGCTCCCGTAT                       G 5 For           ATCCGTCACACCTGCTCTTGAGTCAAAGAGTTTAGGGAGGAGCTA           ACATAACAGTGGTGTTGGCTCCCGTAT                       G 7 For           ATCCGTCACACCTGCTCTAACAACAATGCATCAGCGGGCTGGGAA           CGCATGCGGTGGTGTTGGCTCCCGTAT                       G 8 For           ATCCGTCACACCTGCTCTGAACAGGTTATAAGCAGGAGTGATAGT           TTCAGGATCTGGTGTTGGCTCCCGTAT                       G 9 For           ATCCGTCACACCTGCTCTCGGCGGCTCGCAAACCGAGTGGTCAGC           ACCCGGGTTGGTGTTGGCTCCCGTAT                       G 10 For           ATCCGTCACACCTGCTCTGCGCAAGACGTAATCCACAAGACCGTG           AAAACATAGTGGTGTTGGCTCCCGTAT            
Glucosamine (from LPS) Reverse Sequences:
 
         [0000]                            G 1 Rev           ATACGGGAGCCAACACCATTCCCCCCTCTCGTTCTGTTGCCCCGT           ATCCTAATTAGAGCAGGTGTGACGGAT                       G 2 Rev           ATACGGGAGCCAACACCAGCCGGATATCCGATGTGCTTGTCTGAC           CTGGTCCGAGAGCAGGTGTGACGGAT                       G 4 Rev           ATACGGGAGCCAACACCATTCCCCCCTCTCGTTCTGTTGCCCCGT           ATCCTAATTAGAGCAGGTGTGACGGAT                       G 5 Rev           ATACGGGAGCCAACACCACTGTTATGTTAGCTCCTCCCTAAACTC           TTTGACTCAAGAGCAGGTGTGACGGAT                       G 7 Rev           ATACGGGAGCCAACACCACCGCATGCGTTCCCAGCCCGCTGATGC           ATTGTTGTTAGAGCAGGTGTGACGGAT                       G 8 Rev           ATACGGGAGCCAACACCAGATCCTGAAACTATCACTCCTGCTTAT           AACCTGTTCAGAGCAGGTGTGACGGAT                       G 9 Rev           ATACGGGAGCCAACACCAACCCGGGTGCTGACCACTCGGTTTGCG           AGCCGCCGAGAGCAGGTGTGACGGAT                       G 10 Rev           ATACGGGAGCCAACACCACTATGTTTTCACGGTCTTGTGGATTAC           GTCTTGCGCAGAGCAGGTGTGACGGAT            
KDO Antigen from LPS (Forward Primed):
 
         [0000]                            K 2 For           ATCCGTCACACCTGCTCTAGGCGTAGTGACTAAGTCGCGCGAAAA           TCACAGCATTGGTGTTGGCTCCCGTAT                       K 5 For           ATCCGTCACACCTGCTCTCAGCGGCAGCTATACAGTGAGAACGGA           CTAGTGCGTTGGTGTTGGCTCCCGTAT                       K 7 For           ATCCGTCACACCTGCTCTGGCAAATAATACTAGCGATGATGGATC           TGGATAGACTGGTGTTGGCTCCCGTAT                       K 8 For           ATCCGTCACACCTGCTCTGGGGGTGCGACTTAGGGTAAGTGGGAA           AGACGATGCTGGTGTTGGCTCCCGTAT                       K 9 For           ATCCGTCACACCTGCTCTCAAGAGGAGATGAACCAATCTTAGTCC           GACAGGCGGTGGTGTTGGCTCCCGTAT                       K 10 For           ATCCGTCACACCTGCTCTGGCCCGGAATTGTCATGACGTCACCTA           CACCTCCTGTGGTGTTGGCTCCCGTAT            
KDO Antigen from LPS (Reverse Primed):
 
         [0000]                            K 2 Rev           ATACGGGAGCCAACACCAATGCTGTGATTTTCGCGCGACTTAGTC           ACTACGCCTAGAGCAGGTGTGACGGAT                       K 5 Rev           ATACGGGAGCCAACACCAACGCACTAGTCCGTTCTCACTGTATAG           CTGCCGCTGAGAGCAGGTGTGACGGAT                       K 7 Rev           ATACGGGAGCCAACACCAGTCTATCCAGATCCATCATCGCTAGTA           TTATTTGCCAGAGCAGGTGTGACGGAT                       K 8 Rev           ATACGGGAGCCAACACCAGCATCGTCTTTCCCACTTACCCTAAGT           CGCACCCCCAGAGCAGGTGTGACGGAT                       K 9 Rev           ATACGGGAGCCAACACCACCGCCTGTCGGACTAAGATTGGTTCAT           CTCCTCTTGAGAGCAGGTGTGACGGAT                       K 10 Rev           ATACGGGAGCCAACACCACAGGAGGTGTAGGTGACGTCATGACAA           TTCCGGGCCAGAGCAGGTGTGACGGAT              Leishmania donovani  Binding Aptamer Sequences:
   Leishmania donovani  Clone: 940-3
 
         [0000]                            Forward:           GATACGGGAGCCAACACCACCCGTATCGTTCCCAATGCACTCAGA           GCAGGTGTGACGGATG                       Reverse:           CATCCGTCACACCTGCTCTGAGTGCATTGGGAACGATACGGGTGG           TGTTGGCTCCCGTATG              Leishmania donovani  Clone: 940-5
 
         [0000]                            Forward:           GATACGGGAGCCAACACCACGTTCCCATACAAGTTACTGACAGAG           CAGGTGTGACGGATG                       Reverse:           CATCCGTCACACCTGCTCTGTCAGTAACTTGTATGGGAACGTGGT           GTTGGCTCCCGTATC            
Whole LPS from  E. coli  O111:B4 Binding Aptamer Sequences (Forward Primed):
 
         [0000]                            LPS 1 For           ATCCGTCACCCCTGCTCTCGTCGCTATGAAGTAACAAAGATAGGA           GCAATCGGGTGGTGTTGGCTCCCGTAT                       LPS 3 For           ATCCGTCACACCTGCTCTAACGAAGACTGAAACCAAAGCAGTGAC           AGTGCTGAATGGTGTTGGCTCCCGTAT                       LPS 4 For           ATCCGTCACACCTGCTCTCGGTGACAATAGCTCGATCAGCCCAAA           GTCGTCAGATGGTGTTGGCTCCCGTAT                       LPS 6 For           ATCCGTCACACCTGCTCTAACGAAATAGACCACAAATCGATACTT           TATGTTATTGGTGTTGGCTCCCGTAT                       LPS 7 For           ATCCGTCACACCTGCTCTGTCGAATGCTCTGCCTGGAAGAGTTGT           TAGCAGGGATGGTGTTGGCTCCCGTAT                       LPS 8 For           ATCCGTCACACCTGCTCTTAAGCCGAGGGGTAAATCTAGGACAGG           GGTCCATGATGGTGTTGGCTCCCGTAT                       LPS 9 For           ATCCGTCACACCTGCTCTACTGGCCGGCTCAGCATGACTAAGAAG           GAAGTTATGTGGTGTTGGCTCCCGTAT                       LPS 10 For           ATCCGTCACACCTGCTCTGGTACGAATCACAGGGGATGCTGGAAG           CTTGGCTCTTGGTGTTGGCTCCCGTAT            
Whole LPS from  E. coli  O111:B4 Binding Aptamer Sequences (Reverse Primed):
 
         [0000]    
       
         
               
               
             
           
               
                   
                 LPS 1 Rev 
               
               
                   
                 ATACGGGAGCCAACACCACCCGATTGCTCCTATCTTTGTTACTTCA 
               
               
                   
                 TAGCGACGAGAGCAGGGGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 3 Rev 
               
               
                   
                 ATACGGGAGCCAACACCATTCAGCACTGTCACTGCTTTGGTTTCAG 
               
               
                   
                 TCTTCGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 4 Rev 
               
               
                   
                 ATACGGGAGCCAACACCATCTGACGACTTTGGGCTGATCGAGCTAT 
               
               
                   
                 TGTCACCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 6 Rev 
               
               
                   
                 ATACGGGAGCCAACACCAATAACATAAAGTATCGATTTGTGGTCTA 
               
               
                   
                 TTTCGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 7 Rev 
               
               
                   
                 ATACGGGAGCCAACACCATCCCTGCTAACAACTCTTCCAGGCAGAG 
               
               
                   
                 CATTCGACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 8 Rev 
               
               
                   
                 ATACGGGAGCCAACACCATCATGGACCCCTGTCCTAGATTTACCCC 
               
               
                   
                 TCGGCTTAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 9 Rev 
               
               
                   
                 ATACGGGAGCCAACACCACATAACTTCCTTCTTAGTCATGCTGAGC 
               
               
                   
                 CGGCCAGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                   
                 LPS 10 Rev 
               
               
                   
                 ATACGGGAGCCAACACCAAGAGCCAAGCTTCCAGCATCCCCTGTGA 
               
               
                   
                 TTCGTACCAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
       Methylphosphonic Acid (MPA) Binding Aptamer Sequences: 
       [0020]      
         [0000]    
       
         
               
               
             
           
               
                   
                 MPA Forward 
               
               
                   
                 ATACGGGAGCCAACACCATTAAATCAATTGTGCCGTGTTCCTCTTG 
               
               
                   
                 TCTCATCGAGAGCAGGTTGTACGGAT 
               
               
                   
                   
               
               
                   
                 MPA Reverse 
               
               
                   
                 ATCCGTACAACCTGCTCTCGATGAGACAAGAGGAACACGGCACAAT 
               
               
                   
                 TGATTTAATGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Malathion Binding Aptamer Sequences: 
       [0021]      
         [0000]    
       
         
               
               
             
           
               
                   
                 M17 Forward 
               
               
                   
                 ATACGGGAGCCAACACCAGCAGTCAAGAAGTTAAGAGAAAAACAAT 
               
               
                   
                 TGTGTATAAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 M17 Reverse 
               
               
                   
                 ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTC 
               
               
                   
                 TTGACTGCTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 M21 Forward 
               
               
                   
                 ATCCGTCACACCTGCTCTGCGCCACAAGATTGCGGAAAGACACCCG 
               
               
                   
                 GGGGGCTTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 M21 Reverse 
               
               
                   
                 ATACGGGAGCCAACACCAAGCCCCCCGGGTGTCTTTCCGCAATCTT 
               
               
                   
                 GTGGCGCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 M25 Forward 
               
               
                   
                 ATCCGTCACACCTGCTCTGGCCTTATGTAAAGCGTTGGGTGGTGTT 
               
               
                   
                 GGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 M25 Reverse 
               
               
                   
                 ATACGGGAGCCAACACCACCCAACGCTTTACATAAGGCCAGAGCAG 
               
               
                   
                 GTGTGACGGAT 
               
             
          
         
       
     
       Poly-D-Glutamic Acid Binding Aptamer Sequences: 
       [0022]      
         [0000]    
       
         
               
               
             
           
               
                   
                 PDGA 2F 
               
               
                   
                 CATCCGTCACACCTGCTCTGGTTCGCCCCGGTCAAGGAGAGTGGTG 
               
               
                   
                 TTGGCTCCCGTATC 
               
               
                   
                   
               
               
                   
                 PDGA 2R 
               
               
                   
                 GATACGGGAGCCAACACCACTCTCCTTGACCGGGGCGAACCAGAGC 
               
               
                   
                 AGGTGTGACGGATG 
               
               
                   
                   
               
               
                   
                 PDGA 5F 
               
               
                   
                 CATCCGTCACACCTGCTCTGGATAAGATCAGCAACAAGTTAGTGGT 
               
               
                   
                 GTTGGCTCCCGTATC 
               
               
                   
                   
               
               
                   
                 PDGA 5R 
               
               
                   
                 GATACGGGAGCCAACACCACTAACTTGTTGCTGATCTTATCAGAGC 
               
               
                   
                 AGGTGTGACGGATG 
               
             
          
         
       
     
       Rough Ra Mutant LPS Core Antigen Binding Aptamer Sequences (Forward Primed): 
       [0023]      
         [0000]    
       
         
               
               
             
           
               
                   
                 R 1F 
               
               
                   
                 ATCCGTCACACCTGCTCTCCGCACGTAGGACCACTTTGGTACACGC 
               
               
                   
                 TCCCGTAGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 R 5F 
               
               
                   
                 ATCCGTCACACCTGCTCTACGGATGAACGAAGATTTTAAAGTCAAG 
               
               
                   
                 CTAATGCATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 R 6F 
               
               
                   
                 ATCCGTCACACCTGCTCTGTAGTGAAGAGTCCGCAGTCCACGCTGT 
               
               
                   
                 TCAACTCATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 R 7F 
               
               
                   
                 ATCCGTCACACCTGCTCTACCGGCTGGCACGGTTATGTGTGACGGG 
               
               
                   
                 CGAAGATATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 R 8F 
               
               
                   
                 ATCCGTCACACCTGCTCTACCGGCTGGCACGGTTATGTGTGACGGG 
               
               
                   
                 CGAAGATATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 R 9F 
               
               
                   
                 ATCCGTCACACCTGCTCTGCGTGTGGAGCGCCTAGGTGAGTGGTGT 
               
               
                   
                 TGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 R 10F 
               
               
                   
                 ATCCGTCACACCTGCTCTGATGTCCCTTTGAAGAGTTCCATGACGC 
               
               
                   
                 TGGCTCCTTGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Rough Ra Mutant LPS Core Antigen Binding Aptamer Sequences (Reverse Primed): 
       [0024]      
         [0000]    
       
         
               
               
             
           
               
                   
                 R 1R 
               
               
                   
                 ATACGGGAGCCAACACCACTACGGGAGCGTGTACCAAAGTGGTCCT 
               
               
                   
                 ACGTGCGGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 R 5R 
               
               
                   
                 ATACGGGAGCCAACACCATGCATTAGCTTGACTTTAAAATCTTCGT 
               
               
                   
                 TCATCCGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 R 6R 
               
               
                   
                 ATACGGGAGCCAACACCATGAGTTGAACAGCGTGGACTGCGGACTC 
               
               
                   
                 TTCACTACAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 R 7R 
               
               
                   
                 ATACGGGAGCCAACACCATATCTTCGCCCGTCACACATAACCGTGC 
               
               
                   
                 CAGCCGGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 R 8R 
               
               
                   
                 ATACGGGAGCCAACACCATATCTTCGCCCGTCACACATAACCGTGC 
               
               
                   
                 CAGCCGGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 R 9R 
               
               
                   
                 ATACGGGAGCCAACACCACTCACCTAGGCGCTCCACACGCAGAGCA 
               
               
                   
                 GGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 R 10R 
               
               
                   
                 ATACGGGAGCCAACACCAAGGAGCCAGCGTCATGGAACTCTTCAAA 
               
               
                   
                 GGGACATCAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
       Soman Binding Aptamer Sequences: 
       [0025]      
         [0000]    
       
         
               
               
             
           
               
                   
                 Soman 20F 
               
               
                   
                 ATACGGGAGCCAACACCATAGTGTTGGGCCAATACGGTAACGTGTC 
               
               
                   
                 CTTGGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 20R 
               
               
                   
                 ATCCGTCACACCTGCTCTCCAAGGACACGTTACCGACGAATTGGCC 
               
               
                   
                 CAACACTATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 23F 
               
               
                   
                 ATACGGGAGCCAACACCACACATACGAGTTATCTCGAGTAGAGCAT 
               
               
                   
                 GTTTTGCCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 23R 
               
               
                   
                 ATCCGTCACACCTGCTCTGGCAAAACATGCTCTACTCGAGATAACT 
               
               
                   
                 CGTATGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 24F 
               
               
                   
                 ATACGGGAGCCAACACCAGGCCATCTATTGTTCGTTTTTCTATTTA 
               
               
                   
                 TCTCACCCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Somna 24R 
               
               
                   
                 ATCCGTCACACCTGCTCTGGGTGAGATAAATAGAAAAACGAACAAT 
               
               
                   
                 AGATGGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 25F 
               
               
                   
                 ATACGGGAGCCAACACCACACATACGAGTTATCTCGAGTAGAGCAT 
               
               
                   
                 GTTTTGCCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 25R 
               
               
                   
                 ATCCGTCACACCTGCTCTGGCAAAACATGCTCTACTCGAGATAACT 
               
               
                   
                 CGTATGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 28F 
               
               
                   
                 ATACGGGAGCCAACACCATCCATAGCTCATCTATACCCTCTTCCGA 
               
               
                   
                 GTCCCACCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 28R 
               
               
                   
                 ATCCGTCACACCTGCTCTGGTGGGACTCGGAAGAGGGTATAGATGA 
               
               
                   
                 GCTATGGATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 33F 
               
               
                   
                 ATACGGGAGCCAACACCAGAGCAGGTGTGACGGATAGTGACGGATG 
               
               
                   
                 CAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 33R 
               
               
                   
                 ATCCGTCACACCTGCTCTGCATCCGTCACTATCCGTCACACCTGCT 
               
               
                   
                 CTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 41F 
               
               
                   
                 ATACGGGAGCCAACACCACCTTATGACGCCTCAGTACCACATCGTT 
               
               
                   
                 TAGTCTGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 41R 
               
               
                   
                 ATCCGTCACACCTGCTCTACAGACTAAACGATGTGGTACTGAGGCG 
               
               
                   
                 TCATAAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 45F 
               
               
                   
                 ATACGGGAGCCAACACCACCCGTTTTTGATCTAATGAGGATACAAT 
               
               
                   
                 ATTCGTCTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 45R 
               
               
                   
                 ATCCGTCACACCTGCTCTAGACGAATATTGTATCCTCATTAGATCA 
               
               
                   
                 AAAACGGGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 46F 
               
               
                   
                 ATACGGGAGCCAACACCATCGAGCTCCTTGGCCCCGTTAGGATTAA 
               
               
                   
                 CGTGATGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 46R 
               
               
                   
                 ATCCGTCACACCTGCTCTACATCACGTTAATCCTAACGGGGCCAAG 
               
               
                   
                 GAGCTCGATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 47F 
               
               
                   
                 ATACGGGAGCCAACACCATCAGAACCAAATATACATCTTCCTATGA 
               
               
                   
                 TATGGTGGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 47R 
               
               
                   
                 ATCCGTCACACCTGCTCTCCACCATATCATAGGAAGATGTATATTT 
               
               
                   
                 GGTTCTGATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 Soman 48F 
               
               
                   
                 ATACGGGAGCCAACACCACACGATTGCTCCTCTCATTGTTACTTCA 
               
               
                   
                 TAGCGACGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 Soman 48R 
               
               
                   
                 ATCCGTCACACCTGCTCTCGTCGCTATGAAGTAACAATGAGAGGAG 
               
               
                   
                 CAATCGTGTGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Teichoic Acid or Lipoteichoic Acid Binding Aptamer Sequences: 
       [0026]      
         [0000]                            T5 F           GATACGGGACGACACCACACTATGGGTCGTTTAGCATCAAGGCTAG           CCAAGCCAGCAGAGGTGTGGTGAATG                       T5 R           CATTCACCACACCTCTGCTGGCTTGGCTAGCCTTGATGCTAAACGA           CCCATAGTGTGGTGTCGTCCCGTATC                       T6 F           CATTCACCACACCTCTGCTGGAGGAGGAAGTGGTCTGGAGTTACTT           GACATAGTGTGGTGTCGTCCCGTATC                       T6 R           GATACGGGACGACACCACACTATGTCAAGTAACTCCAGACCACTTC           CTCCTCCAGCAGAGGTGTGGTGAATG                       T7 F           CATTCACCACACCTCTGCTGGACGGAAACAATCCCCGGGTACGAGA           ATCAGGGTGTGGTGTCGTCCCGTATC                       T7 R           GATACGGGACGACACCACACCCTGATTCTCGTACCCGGGGATTGTT           TCCGTCCAGCAGAGGTGTGGTGAATG                       T9 F           CATTCACCACACCTCTGCTGGAAACCTACCATTAATGAGACATGAT           GCGGTGGTGTGGTGTCGTCCCGTATC                       T9 R           GATACGGGACGACACCACACCACCGCATCATGTCTCATTAATGGTA           GGTTTCCAGCAGAGGTGTGGTGAATG              E. coli  O157 Lipopolysaccharide (LPS)
 
         [0000]                            E-5F           ATCCGTCACACCTGCTCTGGTGGAATGGACTAAGCTAGCTAGCGTT           TTAAAAGGTGGTGTTGGCTCCCGTAT                       E-11F           ATCCGTCACACCTGCTCTGTAAGGGGGGGGAATCGCTTTCGTCTTA           AGATGACATGGTGTTGGCTCCCGTAT                       E-12F           ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTG           TTGGCTCCCGTAT(59)                       E-16F           ATCCGTCACACCTGCTCTATCCGTCACGCCTGCTCTATCCGTCACA           CCTGCTCTGGTGTTGGCTCCCGTAT                       E-17F           ATCCGTCACACCTGCTCTATCAAATGTGCAGATATCAAGACGATTT           GTACAAGATGGTGTTGGCTCCCGTAT                       E-18F           ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAA           CGATAGAATGGTGTTGGCTCCCGTAT                       E-19F           ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAA           CGATAGAATGGTGTTGGCTCCCGTAT                       E-5R           ATACGGGAGCCAACACCACCTTTTAAAACGCTAGCTAGCTTAGTCC           ATTCCACCAGAGCAGGTGTGACGGAT                       E-11R           ATACGGGAGCCAACACCATGTCATCTTAAGACGAAAGCGATTCCCC           CCCCTTACAGAGCAGGTGTGACGGAT                       E-12R           ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGC           AGGTGTGACGGAT                       E-16R           ATACGGGAGCCAACACCAGAGCAGGTGTGACGGATAGAGCAGGCGT           GACGGATAGAGCAGGTGTGACGGAT                       E-17R           ATACGGGAGCCAACACCATCTTGTACAAATCGTCTTGATATCTGCA           CATTTGATAGAGCAGGTGTGACGGAT                       E-18R           ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTG           CCATCTACAGAGCAGGTGTGACGGAT                       E-19R           ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTG           CCATCTACAGAGCAGGTGTGACGGAT            
Listeriolysin (a Surface Protein on  Listeria monocytogenes )
 
         [0000]    
       
         
               
               
             
           
               
                   
                 LO-10F 
               
               
                   
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTG 
               
               
                   
                 TTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-11F 
               
               
                   
                 ATCCGTCACACCTGCTCTGGTGGAATGGACTAAGCTAGCTAGCGTT 
               
               
                   
                 TTAAAAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-13F 
               
               
                   
                 ATCCGTCACACCTGCTCTTAAAGTAGAGGCTGTTCTCCAGACGTCG 
               
               
                   
                 CAGGAGGATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-15F 
               
               
                   
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAA 
               
               
                   
                 CGATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-16F 
               
               
                   
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAA 
               
               
                   
                 CGATAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-17F 
               
               
                   
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGC 
               
               
                   
                 AGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-19F 
               
               
                   
                 ATCCGTCACACCTGCTCTTGGGCAGGAGCGAGAGACTCTAATGGTA 
               
               
                   
                 AGCAAGAATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-20F 
               
               
                   
                 ATCCGTCACACCTGCTCTCCAACAAGGCGACCGACCGCATGCAGAT 
               
               
                   
                 AGCCAGGTTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-10R 
               
               
                   
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGC 
               
               
                   
                 AGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-11R 
               
               
                   
                 ATACGGGAGCCAACACCACCTTTTAAAACGCTAGCTAGCTTAGTCC 
               
               
                   
                 ATTCCACCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-13R 
               
               
                   
                 ATACGGGAGCCAACACCATCCTCCTGCGACGTCTGGAGAACAGCCT 
               
               
                   
                 CTACTTTAAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-15R 
               
               
                   
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTG 
               
               
                   
                 CCATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-16R 
               
               
                   
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTG 
               
               
                   
                 CCATCTACAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-17R 
               
               
                   
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTG 
               
               
                   
                 TTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 LO-19R 
               
               
                   
                 ATACGGGAGCCAACACCATTCTTGCTTACCATTAGAGTCTCTCGCT 
               
               
                   
                 CCTGCCCAAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 LO-20R 
               
               
                   
                 ATACGGGAGCCAACACCAACCTGGCTATCTGCATGCGGTCGGTCGC 
               
               
                   
                 CTTGTTGGAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
       Listeriolysin (Alternate Form of  Listeria  Surface Protein Designated “Pest-Free”) 
       [0027]      
         [0000]                            LP-3F           ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAA           CGATAGAATGGTGTTGGCTCCCGTAT                       LP-11F           ATCCGTCACACCTGCTCTAACCAAAAGGGTAGGAGACCAAGCTAGC           GATTTGGATGGTGTTGGCTCCCGTAT                       LP-13F           ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTG           TTGGCTCCCGTAT                       LP-14F           ATCCGTCACACCTGCTCTGAAGCCTAACGGAGAAGATGGCCCTACT           GCCGTAGGTGGTGTTGGCTCCCGTAT                       LP-15F           ATCCGTCACACCTGCTCTACTAAACAAGGGCAAACTGTAAACACAG           TAGGGGCGTGGTGTTGGCTCCCGTAT                       LP-17F           ATCCGTCACACCTGCTCTGGTGTTGGCTCCCGTATAGCTTGGCTCC           CGTATGGTGTTGGCTCCCGTAT                       LP-18F           ATCCGTCACACCTGCTCTGTCGCGATGATGAGCAGCAGCGCAGGAG           GGAGGGGGTGGTGTTGGCTCCCGTAT                       LP-20F           ATCCGTCACACCTGCTCTGATCAGGGAAGACGCCAACACTGGTGTT           GGCTCCCGTAT                       LP-3R           ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTG           CCATCTACAGAGCAGGTGTGACGGAT                       LP-11R           ATACGGGAGCCAACACCATCCAAATCGCTAGCTTGGTCTCCTACCC           TTTTGGTTAGAGCAGGTGTGACGGAT                       LP-13R           ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGC           AGGTGTGACGGAT                       LP-14R           ATACGGGAGCCAACACCACCTACGGCAGTAGGGCCATCTTCTCCGT           TAGGCTTCAGAGCAGGTGTGACGGAT                       LP-15R           ATACGGGAGCCAACACCACGCCCCTACTGTGTTTACAGTTTGCCCT           TGTTTAGTAGAGCAGGTGTGACGGAT                       LP-17R           ATACGGGAGCCAACACCATACGGGAGCCAAGCTATACGGGAGCCAA           CACCAGAGCAGGTGTGACGGAT                       LP-18R           ATACGGGAGCCAACACCACCCCCTCCCTCCTGCGCTGCTGCTCATC           ATCGCGACAGAGCAGGTGTGACGGAT                       LP-20R           ATACGGGAGCCAACACCAGTGTTGGCGTCTTCCCTGATCAGAGCAG           GTGTGACGGAT              Salmonella typhimurium  Lipopolysaccharide (LPS)
 
         [0000]    
       
         
               
               
             
           
               
                   
                 St-7F 
               
               
                   
                 ATCCGTCACACCTGCTCTGTCCAAAGGCTACGCGTTAACGTGGTGT 
               
               
                   
                 TGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-10F 
               
               
                   
                 ATCCGTCACACCTGCTCTGGAGCAATATGGTGGAGAAACGTGGTGT 
               
               
                   
                 TGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-11F 
               
               
                   
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTG 
               
               
                   
                 TTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-15F 
               
               
                   
                 ATCCGTCACACCTGCTCTGAACAGGATAGGGATTAGCGAGTCAACT 
               
               
                   
                 AAGCAGCATGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-16F 
               
               
                   
                 ATCCGTCACACCTGCTCTGGCGGACAGGAAATAAGAATGAACGCAA 
               
               
                   
                 AATTTATCTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-18F 
               
               
                   
                 ATCCGTCACACCTGCTCTACGCAACGCGACAGGAACATTCATTATA 
               
               
                   
                 GAATGTGTTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-19F 
               
               
                   
                 ATCCGTCACACCTGCTCTCGGCTGCAATGCGGGAGAGTAGGGGGGA 
               
               
                   
                 ACCAAACCTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-20F 
               
               
                   
                 ATCCGTCACACCTGCTCTATGACTGGAACACGGGTATCGATGATTA 
               
               
                   
                 GATGTCCTTGGTGTTGGCTCCCGTAT 
               
               
                   
                   
               
               
                   
                 St-7R 
               
               
                   
                 ATACGGGAGCCAACACCACGTTAACGCGTAGCCTTTGGACAGAGCA 
               
               
                   
                 GGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-10R 
               
               
                   
                 ATACGGGAGCCAACACCACGTTTCTCCACCATATTGCTCCAGAGCA 
               
               
                   
                 GGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-11R 
               
               
                   
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGC 
               
               
                   
                 AGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-15R 
               
               
                   
                 ATACGGGAGCCAACACCATGCTGCTTAGTTGACTCGCTAATCCCTA 
               
               
                   
                 TCCTGTTCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-16R 
               
               
                   
                 ATACGGGAGCCAACACCAGATAAATTTTGCGTTCATTCTTATTTCC 
               
               
                   
                 TGTCCGCCAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-18R 
               
               
                   
                 ATACGGGAGCCAACACCAACACATTCTATAATGAATGTTCCTGTCG 
               
               
                   
                 CGTTGCGTAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-19R 
               
               
                   
                 ATACGGGAGCCAACACCAGGTTTGGTTCCCCCCTACTCTCCCGCAT 
               
               
                   
                 TGCAGCCGAGAGCAGGTGTGACGGAT 
               
               
                   
                   
               
               
                   
                 St-20R 
               
               
                   
                 ATACGGGAGCCAACACCAAGGACATCTAATCATCGATACCCGTGTT 
               
               
                   
                 CCAGTCATAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0028]      FIG. 1 . is a schematic illustration that illustrates a comparison of possible nucleic acid FRET assay formats. 
           [0029]      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. 
           [0030]    FIGS.  3 A.,  3 B., and  3 C 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. 
           [0031]      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. 
           [0032]    FIGS.  5 A.,  5 B,  5 C, and  5 D. 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. 
           [0033]      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). 
           [0034]      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 
       [0035]    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. 
         [0036]    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. 
         [0037]    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. 
         [0038]    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. 
         [0039]    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. 
         [0040]    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. 
         [0041]    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. 
         [0042]    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. 
         [0043]    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. 
         [0044]    The final FRET aptamers are able to act as one-step “lights on” or “lights off” binding and detection components in assays. 
         [0045]    Intrachain FRET-aptamers that are to be used in assays with long shelf-lives may be lyophilized (freeze-dried) and reconstituted. 
         [0046]      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. 
         [0047]    FIGS.  3 A.,  3 B., and  3 C. 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. 
         [0048]      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. 
         [0049]    FIGS.  5 A.,  5 B.,  5 C., and  5 D. 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. 
         [0050]      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. 
         [0051]      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 
     Competitive Aptamer-FRET Assay for Surface Proteins Extracted from Bacteria ( L. donovani ) 
       [0052]    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 88-91. 
       Example 2 
     Competitive FRET-Aptamer Assay for  E. coli  in Environmental Water Samples or Foods Using LPS Component Aptamers 
       [0053]      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 ,  3 B, and  3 C. 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. 92-107. 
       Example 3 
     Competitive FRET-Aptamer Assay for Enterococci in Environmental Water Samples 
       [0054]    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. 156-163. 
       Example 4 
     Detection of Foot-and-Mouth (FMD) Disease or Other Highly Communicable Viruses Among Animal or Human Populations 
       [0055]    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 VP 1 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 with SEQ ID NO&#39;s 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.  5 A.,  5 B,  5 C. and  5 D. Candidate DNA aptamer sequences for detection of the FMD peptide and associated strains of FMD virus are given in SEQ ID Nos. 200-213. 
       Example 5 
     Detection of Organophosphorus (OP) Nerve Agent, Pesticides, and Acetylcholine (ACh) 
       [0056]    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. 1-26, 48-59, 108-115, and 134-155. Amino-MPA and para-aminophenyl-soman were immobilized on tosyl-magnetic beads and used for aptamer selection. 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. 
         [0057]    Other potential examples of uses for competitive FRET-aptamer assays include, but are not limited to: 
         [0000]    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. 27-36), 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. 27-36) and  Clostridium botulinum  in vivo.
 
3) Detection and quantitation of  Campylobacter jejuni  and related  Campylobacter  species (SEQ ID Nos. 42-47) in foods and water to prevent foodborne or waterborne illness outbreaks in a 2006 JCLA paper.
 
4) Detection and quantitation of poly-D-glutamic acid (PDGA; SEQ ID Nos. 116-119) 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.
 
         [0058]    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.