Patent 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.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of copending U.S. application Ser. No. 11/433,283 filed on May 12, 2006, which is incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to the field of aptamer- and nucleic acid-based diagnostics. More particularly, it relates to methods for the production and use of fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers for competitive displacement aptamer assay formats. The present invention provides for aptamer-related FRET assay schemes involving competitive displacement formats in which the aptamer contains fluorophores (“F”) (is F-labeled) and the target contains quenchers (“Q”) (is Q-labeled), or vice versa. The aptamer can be F-labeled or Q-labeled by incorporation of the F or Q derivatives of nucleotide triphosphates. Incorporation may be accomplished by simple chemical conjugations through bifunctional linkers, or key functional groups such as aldehydes, carbodiimides, carboxyls, N-hydroxy-succinimide (NHS) esters, thiols, etc. 
         [0004]    2. Background Information 
         [0005]    Competitive displacement aptamer FRET is a new class of assay desirable for its use in rapid (within minutes), one-step, homogeneous assays involving no wash steps (simple bind and detect quantitative assays). Others have described FRET-aptamer methods for various target analytes that consist of placing the F and Q moieties either on the 5′ and 3′ ends respectively to act like a “molecular (aptamer) beacon” or placing only F in the heart of the aptamer structure to be “quenched” by another proximal F or the DNA or RNA itself. These preceding FRET-aptamer methods are all highly engineered and based on some prior knowledge of particular aptamer sequences and secondary structures, thereby enabling clues as to where F might be placed in order to optimize FRET results. 
       SUMMARY OF THE INVENTION 
       [0006]    The nucleic acid-based “molecular beacons” snap open upon binding to an analyte or upon hybridizing to a complementary sequence, but beacons have always been end-labeled with F and Q at the 3′ and 5′ ends. The present invention provides that F-labeled or Q-labeled aptamers may be labeled anywhere in their structure that places the F or Q within the Förster distance of approximately 60-85 Angstroms of the corresponding F or Q on the labeled target analyte to achieve quenching prior to or after target analyte binding to the aptamer “binding pocket” (typically a “loop” in the secondary structure). The F and Q molecules used can include any number of appropriate fluorophores and quenchers as long as they are spectrally matched so the emission spectrum of F overlaps significantly (almost completely) with the absorption spectrum of Q. 
         [0007]    A process in which F and Q are incorporated into an aptamer population is generally referred to as “doping.” The present invention provides a new method for natural selection of F-labeled or Q-labeled aptamers that contain F-NTPs or Q-NTPs in the heart of an aptamer binding loop or pocket by PCR, asymmetric PCR (using a 100:1 forward:reverse primer ratio), or other enzymatic means. The present invention describes a strain of aptamer in which F and Q are incorporated into an aptamer population via their nucleotide triphosphate derivatives (for example, Alexfluor™-NTP&#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. The invention described herein may use one or more of the following aptamer sequences (the following aptamer sequences are collectively referred to as the “SEQ Aptamers.”) (The SEQ Aptamer identifiers are arranged alphabetically by aptamer target, and are listed 5′ to 3′ from left to right.): 
       Acetylcholine (ACh) Aptamer Sequences: 
       [0014]      
         [0000]    
       
         
               
               
             
           
               
                 ACh1a For 
                   
               
               
                 ATACGGGAGCCAACACCACGATACCCGCTTATGAATTTTAAATTAATTGT 
               
               
                   
               
               
                 GATCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh1a Rev 
               
               
                 ATCCGTCACACCTGCTCTGATCACAATTAATTTAAAATTCATAAGCGGGT 
               
               
                   
               
               
                 ATCGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 1b For 
               
               
                 ATACGGGAGCCAACACCAACTTTCACACATACTTGTTATACCACACGATC 
               
               
                   
               
               
                 TTTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 1b Rev 
               
               
                 ATCCGTCACACCTGCTCTAAAAGATCGTGTGGTATAACAAGTATGTGTGA 
               
               
                   
               
               
                 AAGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 2 For 
               
               
                 ATACGGGAGCCAACACCACTTTGTAACTCATTTGTAGTTTGGGTTGCTCC 
               
               
                   
               
               
                 CCCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 2 Rev 
               
               
                 ATCCGTCACACCTGCTCTAGGGGGAGCAACCCAAACTACAAATGAGTTAC 
               
               
                   
               
               
                 AAAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 3 For 
               
               
                 ATACGGGAGCCAACACCATTTCCCGCTTATCTTCATCCACTGCTTAGCAT 
               
               
                   
               
               
                 ATGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 3 Rev 
               
               
                 ATCCGTCACACCTGCTCTACATATGCTAAGCAGTGGATGAAGATAAGCGG 
               
               
                   
               
               
                 GAAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 5 For 
               
               
                 ATACGGGAGCCAACACCAGGCACTGTATCACACCGTCAAGAATGTGATCC 
               
               
                   
               
               
                 CCTGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 5 Rev 
               
               
                 ATCCGTCACACCTGCTCTCAGGGGATCACATTCTTGACGGTGTGATACAG 
               
               
                   
               
               
                 TGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 6 For 
               
               
                 ATACGGGAGCCAACACCATGTCATTTACCTTCATCATGACAGTGTTAGTA 
               
               
                   
               
               
                 TACGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 6Rev 
               
               
                 ATCCGTCACACCTGCTCTAGGGGATCAAAGCTATGCGACCATGCGAGTGG 
               
               
                   
               
               
                 ATACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 7 For 
               
               
                 ATACGGGAGCCAACACCAGTTGCCGCCTACCTTGATTATTCTACATTACC 
               
               
                   
               
               
                 CGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 7 Rev 
               
               
                 ATCCGTCACACCTGCTCTAACGGGTAATGTAGAATAATCAAGGTAGGCGG 
               
               
                   
               
               
                 CAACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 8 For 
               
               
                 ATACGGGAGCCAACACCAGTATACATACGAAGAGTTGAAACCAATGCTTC 
               
               
                   
               
               
                 GTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 8 Rev 
               
               
                 ATCCGTCACACCTGCTCTGAACGAAGCATTGGTTTCAACTCTTCGTATGT 
               
               
                   
               
               
                 ATACTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 9 For 
               
               
                 ATACGGGAGCCAACACCATACCCCGAATGGCTGTTTTCAGTACCAATATG 
               
               
                   
               
               
                 ACTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 9 Rev 
               
               
                 ATCCGTCACACCTGCTCTGAGTCATATTGGTACTGAAAACAGCCATTCGG 
               
               
                   
               
               
                 GGTATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 10 For 
               
               
                 ATACGGGAGCCAACACCACTGTCACGATCGTCGTTCCTTTTAATCCTTGT 
               
               
                   
               
               
                 GTCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 10 Rev 
               
               
                 ATCCGTCACACCTGCTCTAGACACAAGGATTAAAAGGAACGACGATCGTG 
               
               
                   
               
               
                 ACAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 11 For 
               
               
                 ATACGGGAGCCAACACCACTGGACACTGACCCTCGCACTAGCTTTCTGAC 
               
               
                   
               
               
                 GGGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 11 Rev 
               
               
                 ATCCGTCACACCTGCTCTACCCGGCCGAAGAATAGTGCTCGGTACTTAGT 
               
               
                   
               
               
                 CGCGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 ACh 12 For 
               
               
                 ATACGGGAGCCAACACCATTTGGACTTTAAATAGTGGACTCCTTCTTTGT 
               
               
                   
               
               
                 CTCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 ACh 12 Rev 
               
               
                 ATCCGTCACACCTGCTCTCGAGACAAAGAAGGAGTCCACTATTTAAAGTC 
               
               
                   
               
               
                 CAAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 A25 For 
               
               
                 ATACGGGAGCCAACACCA-TCATTTGCAAATATGAATTCCACTTAAAGAA 
               
               
                   
               
               
                 ATTCA-AGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 A25 Rev 
               
               
                 ATCCGTCACACCTGCTCTTGAATTTCTTTAAGTGGAATTCATATTTGCAA 
               
               
                   
               
               
                 ATGATGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Acyl Homoserine Lactone (AHL) Quorum Sensing Molecules (N-Decanoyl-DL-Homoserine Lactone) 
       [0015]      
         [0000]                        Dec AHL 1For           ATACGGGAGCCAACACCATCCTAACTGGTCTAATTTTTGCTGTTACCGAT               CCCGAGAGCAGGTGTGACGGAT               Dec AHL 1 Rev       ATCCGTCACTCCTGCTCTCGGGATCGGTAACAGCAAAAATTAGACCAGTT               AGGATGGTGTTGGCTCCCGTAT               Dec AHL 13 For       ATACGGGAGCCAACACCAGCCTGACGAAAAAATTTTATCACTAAGTGATA               CGCAAGAGCAGGTGTGACGGAT               Dec AHL 13 Rev       ATCCGTCACACCTGCTCTTGCGTATCACTTAGTGATAAAATTTTTTCGTC               AGGCTGGTGTTGGCTCCCGTAT               Dec AHL 14 For       ATACGGGAGCCAACACCAGACCTACTTCAGAAACGGAAATGTTCTTAGCC               GTCAGAGCAGGTGTGACGGAT               Dec AHL 14 Rev       ATCCGTCACACCTGCTCTGACGGCTAAGAACATTTCCGTTTCTGAAGTAG               GTCTGGTGTTGGCTCCCGTAT               Dec AHL 15 For       ATACGGGAGCCAACACCAGGCCAACGAAACTCCTACTACATATAATGCTT               ATGCAGAGCAGGTGTGACGGAT               Dec AHL 15 Rev       ATCCGTCACACCTGCTCTGCATAAGCATTATATGTAGTAGGAGTTTCGTT               GGCCTGGTGTTGGCTCCCGTAT               Dec AHL 17 For       ATACGGGAGCCAACACCATCCTAACTGGTCTAATTTTTGCTGTTACCGAT               CCCGAGAGCAGGTGTGACGGAT               Dec AHL 17 Rev       ATCCGTCACACCTGCTCTCGGGATCGGTAACAGCAAAAATTAGACCAGTT               AGGATGGTGTTGGCTCCCGTAT              Bacillus thuringiensis  Spore Aptamer Sequence:
 
         [0000]    
       
         
               
               
             
           
               
                 CATCCGTCACACCTGCTCTGGCCACTAACATGGGGACCAGGTGGTGTTGG 
                   
               
               
                   
               
               
                 CTCCCGTATC 
               
             
          
         
       
     
       Botulinum Toxin (BoNT Type A) Aptamer Sequences: 
     BoNT A Holotoxin (Heavy Chain Plus Light Chain Linked Together) 
       [0016]      
         [0000]    
       
         
               
               
             
           
               
                 CATCCGTCACACCTGCTCTGCTATCACATGCCTGCTGAAGTGGTGTTGGC 
                   
               
               
                   
               
               
                 TCCCGTATCA 
               
             
          
         
       
     
       BoNT A 50 kd Enzymatic Light Chain 
       [0017]      
         [0000]                        BoNT A Light Chain 1           CATCCGTCACACCTGCTCTGGGGATGTGTGGTGTTGGCTCCCGTATCAAG               GGCGAATTCT               BoNT A Light Chain 2       CATCCGTCACACCTGCTCTGATCAGGGAAGACGCCAACACGTGGTGTTGG               CTCCCGTATCA               BoNT A Light Chain 3       CATCCGTCACACCTGCTCTGGGTGGTGTTGGCTCCCGTATCAAGGGCGAA               TTCTGCAGATA              Campylobacter jejuni  Binding Aptamers:
 
         [0000]    
       
         
               
               
             
           
               
                 C1 
                   
               
               
                 CATCCGTCACACCTGCTCTGGGGAGGGTGGCGCCCGTCTCGGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
               
                   
               
               
                 C2 
               
               
                 CATCCGTCACACCTGCTCTGGGATAGGGTCTCGTGCTAGATGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
               
                   
               
               
                 C3 
               
               
                 CATCCGTCACACCTGCTCTGGACCGGCGCTTATTCCTGCTTGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
               
                   
               
               
                 C4 
               
               
                 CATCCGTCACACCTGCYCTGGAGCTGATATTGGATGGTCCGGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
               
                   
               
               
                 C5 
               
               
                 CATCCGTCACACCTGCYCYGCCCAGAGCAGGTGTGACGGATGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
               
                   
               
               
                 C6 
               
               
                 CATCCGTCACACCTGCYCYGCCGGACCATCCAATATCAGCTGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATCA 
               
             
          
         
       
     
       Diazinon Binding Aptamers 
       [0018]      
         [0000]                        D12 Forward           ATACGGGAGCCAACACCATTAAATCAATTGTGCCGTGTTGGTCTTGTCTC               ATCGAGAGCAGGTGTGACGGAT               D12 Reverse       ATCCGTCACACCTGCTCTCGATGAGACAAGACCAACACGGCACAATTGAT               TTAATGGTGTTGGCTCCCGTAT               D17 Forward       ATACGGGAGCCAACACCATTTTTATTATCGGTATGATCCTACGAGTTCCT               CCCAAGAGCAGGTGTGACGGAT               D17 Reverse       ATCCGTCACACCTGCTCTTGGGAGGAACTCGTAGGATCATACCGATAATA               AAAATGGTGTTGGCTCCCGTAT               D18 Forward       ATACGGGAGCCAACACCACCGTATATCTTATTATGCACAGCATCACGAAA               GTGCAGAGCAGGTGTGACGGAT               D18 Reverse       ATCCGTCACACCTGCTCTGCACTTTCGTGATGCTGTGCATAATAAGATAT               ACGGTGGTGTTGGCTCCCGTAT               D19 Forward       ATACGGGAGCCAACACCATTAACGTTAAGCGGCCTCACTTCTTTTAATCC               TTTCAGAGCAGGTGTGACGGAT               D19 Reverse       ATCCGTCACACCTGCTCTGAAAGGATTAAAAGAAGTGAGGCCGCTTAACG               TTAATGGTGTTGGCTCCCGTAT               D20 Forward       ATCCGTCACACCTGCTCTAATATAGAGGTATTGCTCTTGGACAAGGTACA               GGGATGGTGTTGGCTCCCGTAT               D20 Reverse       ATACGGGAGCCAACACCATCCCTGTACCTTGTCCAAGAGCAATACCTCTA               TATTAGAGCAGGTGTGACGGAT               D25 Forward       ATACGGGAGCCAACACCATTAACGTTAAGCGGCCTCACTTCTTTTAATCC               TTTCAGAGCAGGTGTGACGGAT               D25 Reverse       ATCCGTCACACCTGCTCTGAAAGGATTAAAAGAAGTGAGGCCGCTTAACG               TTAATGGTGTTGGCTCCCGTAT            
Glucosamine (from LPS) Forward Aptamer Sequences:
 
         [0000]                        G 1 For           ATCCGTCACACCTGCTCTAATTAGGATACGGGGCAACAGAACGAGAGGGG               GGAATGGTGTTGGCTCCCGTAT               G 2 For       ATCCGTCACACCTGCTCTCGGACCAGGTCAGACAAGCACATCGGATATCC               GGCTGGTGTTGGCTCCCGTAT               G 4 For       ATCCGTCACACCTGCTCTAATTAGGATACGGGGCAACAGAACGAGAGGGG               GGAATGGTGTTGGCTCCCGTAT               G 5 For       ATCCGTCACACCTGCTCTTGAGTCAAAGAGTTTAGGGAGGAGCTAACATA               ACAGTGGTGTTGGCTCCCGTAT               G 7 For       ATCCGTCACACCTGCTCTAACAACAATGCATCAGCGGGCTGGGAACGCAT               GCGGTGGTGTTGGCTCCCGTAT               G 8 For       ATCCGTCACACCTGCTCTGAACAGGTTATAAGCAGGAGTGATAGTTTCAG               GATCTGGTGTTGGCTCCCGTAT               G 9 For       ATCCGTCACACCTGCTCTCGGCGGCTCGCAAACCGAGTGGTCAGCACCCG               GGTTGGTGTTGGCTCCCGTAT               G 10 For       ATCCGTCACACCTGCTCTGCGCAAGACGTAATCCACAAGACCGTGAAAAC               ATAGTGGTGTTGGCTCCCGTAT            
Glucosamine (from LPS) Reverse Sequences:
 
         [0000]                        G 1 Rev           ATACGGGAGCCAACACCATTCCCCCCTCTCGTTCTGTTGCCCCGTATCCT               AATTAGAGCAGGTGTGACGGAT               G 2 Rev       ATACGGGAGCCAACACCAGCCGGATATCCGATGTGCTTGTCTGACCTGGT               CCGAGAGCAGGTGTGACGGAT               G 4 Rev       ATACGGGAGCCAACACCATTCCCCCCTCTCGTTCTGTTGCCCCGTATCCT               AATTAGAGCAGGTGTGACGGAT               G 5 Rev       ATACGGGAGCCAACACCACTGTTATGTTAGCTCCTCCCTAAACTCTTTGA               CTCAAGAGCAGGTGTGACGGAT               G 7 Rev       ATACGGGAGCCAACACCACCGCATGCGTTCCCAGCCCGCTGATGCATTGT               TGTTAGAGCAGGTGTGACGGAT               G 8 Rev       ATACGGGAGCCAACACCAGATCCTGAAACTATCACTCCTGCTTATAACCT               GTTCAGAGCAGGTGTGACGGAT               G 9 Rev       ATACGGGAGCCAACACCAACCCGGGTGCTGACCACTCGGTTTGCGAGCCG               CCGAGAGCAGGTGTGACGGAT               G 10 Rev       ATACGGGAGCCAACACCACTATGTTTTCACGGTCTTGTGGATTACGTCTT               GCGCAGAGCAGGTGTGACGGAT            
KDO Antigen from LPS (Forward Primed):
 
         [0000]                        K 2 For           ATCCGTCACACCTGCTCTAGGCGTAGTGACTAAGTCGCGCGAAAATCACA               GCATTGGTGTTGGCTCCCGTAT               K 5 For       ATCCGTCACACCTGCTCTCAGCGGCAGCTATACAGTGAGAACGGACTAGT               GCGTTGGTGTTGGCTCCCGTAT               K 7 For       ATCCGTCACACCTGCTCTGGCAAATAATACTAGCGATGATGGATCTGGAT               AGACTGGTGTTGGCTCCCGTAT               K 8 For       ATCCGTCACACCTGCTCTGGGGGTGCGACTTAGGGTAAGTGGGAAAGACG               ATGCTGGTGTTGGCTCCCGTAT               K 9 For       ATCCGTCACACCTGCTCTCAAGAGGAGATGAACCAATCTTAGTCCGACAG               GCGGTGGTGTTGGCTCCCGTAT               K 10 For       ATCCGTCACACCTGCTCTGGCCCGGAATTGTCATGACGTCACCTACACCT               CCTGTGGTGTTGGCTCCCGTAT            
KDO Antigen from LPS (Reverse Primed):
 
         [0000]                        K 2 Rev           ATACGGGAGCCAACACCAATGCTGTGATTTTCGCGCGACTTAGTCACTAC               GCCTAGAGCAGGTGTGACGGAT               K 5 Rev       ATACGGGAGCCAACACCAACGCACTAGTCCGTTCTCACTGTATAGCTGCC               GCTGAGAGCAGGTGTGACGGAT               K 7 Rev       ATACGGGAGCCAACACCAGTCTATCCAGATCCATCATCGCTAGTATTATT               TGCCAGAGCAGGTGTGACGGAT               K 8 Rev       ATACGGGAGCCAACACCAGCATCGTCTTTCCCACTTACCCTAAGTCGCAC               CCCCAGAGCAGGTGTGACGGAT               K 9 Rev       ATACGGGAGCCAACACCACCGCCTGTCGGACTAAGATTGGTTCATCTCCT               CTTGAGAGCAGGTGTGACGGAT               K 10 Rev       ATACGGGAGCCAACACCACAGGAGGTGTAGGTGACGTCATGACAATTCCG               GGCCAGAGCAGGTGTGACGGAT              Leishmania donovani  Binding Aptamer Sequences:
   Leishmania donovani  Clone: 940-3
 
         [0000]                        Forward:           GATACGGGAGCCAACACCACCCGTATCGTTCCCAATGCACTCAGAGCAGG               TGTGACGGATG               Reverse:       CATCCGTCACACCTGCTCTGAGTGCATTGGGAACGATACGGGTGGTGTTG               GCTCCCGTATG              Leishmania donovani  Clone: 940-5
 
         [0000]                        Forward:           GATACGGGAGCCAACACCACGTTCCCATACAAGTTACTGACAGAGCAGGT               GTGACGGATG               Reverse:       CATCCGTCACACCTGCTCTGTCAGTAACTTGTATGGGAACGTGGTGTTGG               CTCCCGTATC            
Whole LPS from  E. coli  O111:B4 Binding Aptamer Sequences (Forward Primed):
 
         [0000]                        LPS 1 For           ATCCGTCACCCCTGCTCTCGTCGCTATGAAGTAACAAAGATAGGAGCAAT               CGGGTGGTGTTGGCTCCCGTAT               LPS 3 For       ATCCGTCACACCTGCTCTAACGAAGACTGAAACCAAAGCAGTGACAGTGC               TGAATGGTGTTGGCTCCCGTAT               LPS 4 For       ATCCGTCACACCTGCTCTCGGTGACAATAGCTCGATCAGCCCAAAGTCGT               CAGATGGTGTTGGCTCCCGTAT               LPS 6 For       ATCCGTCACACCTGCTCTAACGAAATAGACCACAAATCGATACTTTATGT               TATTGGTGTTGGCTCCCGTAT               LPS 7 For       ATCCGTCACACCTGCTCTGTCGAATGCTCTGCCTGGAAGAGTTGTTAGCA               GGGATGGTGTTGGCTCCCGTAT               LPS 8 For       ATCCGTCACACCTGCTCTTAAGCCGAGGGGTAAATCTAGGACAGGGGTCC               ATGATGGTGTTGGCTCCCGTAT               LPS 9 For       ATCCGTCACACCTGCTCTACTGGCCGGCTCAGCATGACTAAGAAGGAAGT               TATGTGGTGTTGGCTCCCGTAT               LPS 10 For       ATCCGTCACACCTGCTCTGGTACGAATCACAGGGGATGCTGGAAGCTTGG               CTCTTGGTGTTGGCTCCCGTAT            
Whole LPS from  E. coli  O111:B4 Binding Aptamer Sequences (Reverse Primed):
 
         [0000]    
       
         
               
               
             
           
               
                 LPS 1 Rev 
                   
               
               
                 ATACGGGAGCCAACACCACCCGATTGCTCCTATCTTTGTTACTTCATAGC 
               
               
                   
               
               
                 GACGAGAGCAGGGGTGACGGAT 
               
               
                   
               
               
                 LPS 3 Rev 
               
               
                 ATACGGGAGCCAACACCATTCAGCACTGTCACTGCTTTGGTTTCAGTCTT 
               
               
                   
               
               
                 CGTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 4 Rev 
               
               
                 ATACGGGAGCCAACACCATCTGACGACTTTGGGCTGATCGAGCTATTGTC 
               
               
                   
               
               
                 ACCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 6 Rev 
               
               
                 ATACGGGAGCCAACACCAATAACATAAAGTATCGATTTGTGGTCTATTTC 
               
               
                   
               
               
                 GTTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 7 Rev 
               
               
                 ATACGGGAGCCAACACCATCCCTGCTAACAACTCTTCCAGGCAGAGCATT 
               
               
                   
               
               
                 CGACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 8 Rev 
               
               
                 ATACGGGAGCCAACACCATCATGGACCCCTGTCCTAGATTTACCCCTCGG 
               
               
                   
               
               
                 CTTAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 9 Rev 
               
               
                 ATACGGGAGCCAACACCACATAACTTCCTTCTTAGTCATGCTGAGCCGGC 
               
               
                   
               
               
                 CAGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LPS 10 Rev 
               
               
                 ATACGGGAGCCAACACCAAGAGCCAAGCTTCCAGCATCCCCTGTGATTCG 
               
               
                   
               
               
                 TACCAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
       Methylphosphonic Acid (MPA) Binding Aptamer Sequences: 
       [0019]      
         [0000]    
       
         
               
               
             
           
               
                 MPA Forward 
                   
               
               
                 ATACGGGAGCCAACACCATTAAATCAATTGTGCCGTGTTCCTCTTGTCTC 
               
               
                   
               
               
                 ATCGAGAGCAGGTTGTACGGAT 
               
               
                   
               
               
                 MPA Reverse 
               
               
                 ATCCGTACAACCTGCTCTCGATGAGACAAGAGGAACACGGCACAATTGAT 
               
               
                   
               
               
                 TTAATGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Malathion Binding Aptamer Sequences: 
       [0020]      
         [0000]    
       
         
               
               
             
           
               
                 M17 Forward 
                   
               
               
                 ATACGGGAGCCAACACCAGCAGTCAAGAAGTTAAGAGAAAAACAATTGTG 
               
               
                   
               
               
                 TATAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 M17 Reverse 
               
               
                 ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGA 
               
               
                   
               
               
                 CTGCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 M21 Forward 
               
               
                 ATCCGTCACACCTGCTCTGCGCCACAAGATTGCGGAAAGACACCCGGGGG 
               
               
                   
               
               
                 GCTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 M21 Reverse 
               
               
                 ATACGGGAGCCAACACCAAGCCCCCCGGGTGTCTTTCCGCAATCTTGTGG 
               
               
                   
               
               
                 CGCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 M25 Forward 
               
               
                 ATCCGTCACACCTGCTCTGGCCTTATGTAAAGCGTTGGGTGGTGTTGGCT 
               
               
                   
               
               
                 CCCGTAT 
               
               
                   
               
               
                 M25 Reverse 
               
               
                 ATACGGGAGCCAACACCACCCAACGCTTTACATAAGGCCAGAGCAGGTGT 
               
               
                   
               
               
                 GACGGAT 
               
             
          
         
       
     
       Poly-D-Glutamic Acid Binding Aptamer Sequences: 
       [0021]      
         [0000]    
       
         
               
               
             
           
               
                 PDGA 2F 
                   
               
               
                 CATCCGTCACACCTGCTCTGGTTCGCCCCGGTCAAGGAGAGTGGTGTTGG 
               
               
                   
               
               
                 CTCCCGTATC 
               
               
                   
               
               
                 PDGA 2R 
               
               
                 GATACGGGAGCCAACACCACTCTCCTTGACCGGGGCGAACCAGAGCAGGT 
               
               
                   
               
               
                 GTGACGGATG 
               
               
                   
               
               
                 PDGA 5F 
               
               
                 CATCCGTCACACCTGCTCTGGATAAGATCAGCAACAAGTTAGTGGTGTTG 
               
               
                   
               
               
                 GCTCCCGTATC 
               
               
                   
               
               
                 PDGA 5R 
               
               
                 GATACGGGAGCCAACACCACTAACTTGTTGCTGATCTTATCAGAGCAGGT 
               
               
                   
               
               
                 GTGACGGATG 
               
             
          
         
       
     
       Rough Ra Mutant LPS Core Antigen Binding Aptamer Sequences (Forward Primed): 
       [0022]      
         [0000]    
       
         
               
               
             
           
               
                 R 1F 
                   
               
               
                 ATCCGTCACACCTGCTCTCCGCACGTAGGACCACTTTGGTACACGCTCCC 
               
               
                   
               
               
                 GTAGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 5F 
               
               
                 ATCCGTCACACCTGCTCTACGGATGAACGAAGATTTTAAAGTCAAGCTAA 
               
               
                   
               
               
                 TGCATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 6F 
               
               
                 ATCCGTCACACCTGCTCTGTAGTGAAGAGTCCGCAGTCCACGCTGTTCAA 
               
               
                   
               
               
                 CTCATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 7F 
               
               
                 ATCCGTCACACCTGCTCTACCGGCTGGCACGGTTATGTGTGACGGGCGAA 
               
               
                   
               
               
                 GATATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 8F 
               
               
                 ATCCGTCACACCTGCTCTACCGGCTGGCACGGTTATGTGTGACGGGCGAA 
               
               
                   
               
               
                 GATATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 R 9F 
               
               
                 ATCCGTCACACCTGCTCTGCGTGTGGAGCGCCTAGGTGAGTGGTGTTGGC 
               
               
                   
               
               
                 TCCCGTAT 
               
               
                   
               
               
                 R 10F 
               
               
                 ATCCGTCACACCTGCTCTGATGTCCCTTTGAAGAGTTCCATGACGCTGGC 
               
               
                   
               
               
                 TCCTTGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Roueh Ra Mutant LPS Core Antigen Binding Aptamer Sequences (Reverse Primed): 
       [0023]      
         [0000]    
       
         
               
               
             
           
               
                 R 1R 
                   
               
               
                 ATACGGGAGCCAACACCACTACGGGAGCGTGTACCAAAGTGGTCCTACGT 
               
               
                   
               
               
                 GCGGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 5R 
               
               
                 ATACGGGAGCCAACACCATGCATTAGCTTGACTTTAAAATCTTCGTTCAT 
               
               
                   
               
               
                 CCGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 6R 
               
               
                 ATACGGGAGCCAACACCATGAGTTGAACAGCGTGGACTGCGGACTCTTCA 
               
               
                   
               
               
                 CTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 7R 
               
               
                 ATACGGGAGCCAACACCATATCTTCGCCCGTCACACATAACCGTGCCAGC 
               
               
                   
               
               
                 CGGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 8R 
               
               
                 ATACGGGAGCCAACACCATATCTTCGCCCGTCACACATAACCGTGCCAGC 
               
               
                   
               
               
                 CGGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 R 9R 
               
               
                 ATACGGGAGCCAACACCACTCACCTAGGCGCTCCACACGCAGAGCAGGTG 
               
               
                   
               
               
                 TGACGGAT 
               
               
                   
               
               
                 R 10R 
               
               
                 ATACGGGAGCCAACACCAAGGAGCCAGCGTCATGGAACTCTTCAAAGGGA 
               
               
                   
               
               
                 CATCAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
       Soman Binding Aptamer Sequences: 
       [0024]      
         [0000]    
       
         
               
               
             
           
               
                 Soman 20F 
                   
               
               
                 ATACGGGAGCCAACACCATAGTGTTGGGCCAATACGGTAACGTGTCCTTG 
               
               
                   
               
               
                 GAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 20R 
               
               
                 ATCCGTCACACCTGCTCTCCAAGGACACGTTACCGACGAATTGGCCCAAC 
               
               
                   
               
               
                 ACTATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 23F 
               
               
                 ATACGGGAGCCAACACCACACATACGAGTTATCTCGAGTAGAGCATGTTT 
               
               
                   
               
               
                 TGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 23R 
               
               
                 ATCCGTCACACCTGCTCTGGCAAAACATGCTCTACTCGAGATAACTCGTA 
               
               
                   
               
               
                 TGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 24F 
               
               
                 ATACGGGAGCCAACACCAGGCCATCTATTGTTCGTTTTTCTATTTATCTC 
               
               
                   
               
               
                 ACCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Somna 24R 
               
               
                 ATCCGTCACACCTGCTCTGGGTGAGATAAATAGAAAAACGAACAATAGAT 
               
               
                   
               
               
                 GGCCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 25F 
               
               
                 ATACGGGAGCCAACACCACACATACGAGTTATCTCGAGTAGAGCATGTTT 
               
               
                   
               
               
                 TGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 25R 
               
               
                 ATCCGTCACACCTGCTCTGGCAAAACATGCTCTACTCGAGATAACTCGTA 
               
               
                   
               
               
                 TGTGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 28F 
               
               
                 ATACGGGAGCCAACACCATCCATAGCTCATCTATACCCTCTTCCGAGTCC 
               
               
                   
               
               
                 CACCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 28R 
               
               
                 ATCCGTCACACCTGCTCTGGTGGGACTCGGAAGAGGGTATAGATGAGCTA 
               
               
                   
               
               
                 TGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 33F 
               
               
                 ATACGGGAGCCAACACCAGAGCAGGTGTGACGGATAGTGACGGATGCAGA 
               
               
                   
               
               
                 GCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 33R 
               
               
                 ATCCGTCACACCTGCTCTGCATCCGTCACTATCCGTCACACCTGCTCTGG 
               
               
                   
               
               
                 TGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 41F 
               
               
                 ATACGGGAGCCAACACCACCTTATGACGCCTCAGTACCACATCGTTTAGT 
               
               
                   
               
               
                 CTGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 41R 
               
               
                 ATCCGTCACACCTGCTCTACAGACTAAACGATGTGGTACTGAGGCGTCAT 
               
               
                   
               
               
                 AAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 45F 
               
               
                 ATACGGGAGCCAACACCACCCGTTTTTGATCTAATGAGGATACAATATTC 
               
               
                   
               
               
                 GTCTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 45R 
               
               
                 ATCCGTCACACCTGCTCTAGACGAATATTGTATCCTCATTAGATCAAAAA 
               
               
                   
               
               
                 CGGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 46F 
               
               
                 ATACGGGAGCCAACACCATCGAGCTCCTTGGCCCCGTTAGGATTAACGTG 
               
               
                   
               
               
                 ATGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 46R 
               
               
                 ATCCGTCACACCTGCTCTACATCACGTTAATCCTAACGGGGCCAAGGAGC 
               
               
                   
               
               
                 TCGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 47F 
               
               
                 ATACGGGAGCCAACACCATCAGAACCAAATATACATCTTCCTATGATATG 
               
               
                   
               
               
                 GTGGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 47R 
               
               
                 ATCCGTCACACCTGCTCTCCACCATATCATAGGAAGATGTATATTTGGTT 
               
               
                   
               
               
                 CTGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 Soman 48F 
               
               
                 ATACGGGAGCCAACACCACACGATTGCTCCTCTCATTGTTACTTCATAGC 
               
               
                   
               
               
                 GACGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 Soman 48R 
               
               
                 ATCCGTCACACCTGCTCTCGTCGCTATGAAGTAACAATGAGAGGAGCAAT 
               
               
                   
               
               
                 CGTGTGGTGTTGGCTCCCGTAT 
               
             
          
         
       
     
       Teichoic Acid or Lipoteichoic Acid Binding Aptamer Sequences: 
       [0025]      
         [0000]                        T5 F           GATACGGGACGACACCACACTATGGGTCGTTTAGCATCAAGGCTAGCCAA               GCCAGCAGAGGTGTGGTGAATG               T5 R       CATTCACCACACCTCTGCTGGCTTGGCTAGCCTTGATGCTAAACGACCCA               TAGTGTGGTGTCGTCCCGTATC               T6 F       CATTCACCACACCTCTGCTGGAGGAGGAAGTGGTCTGGAGTTACTTGACA               TAGTGTGGTGTCGTCCCGTATC               T6 R       GATACGGGACGACACCACACTATGTCAAGTAACTCCAGACCACTTCCTCC               TCCAGCAGAGGTGTGGTGAATG               T7 F       CATTCACCACACCTCTGCTGGACGGAAACAATCCCCGGGTACGAGAATCA               GGGTGTGGTGTCGTCCCGTATC               T7 R       GATACGGGACGACACCACACCCTGATTCTCGTACCCGGGGATTGTTTCCG               TCCAGCAGAGGTGTGGTGAATG               T9 F       CATTCACCACACCTCTGCTGGAAACCTACCATTAATGAGACATGATGCGG               TGGTGTGGTGTCGTCCCGTATC               T9 R       GATACGGGACGACACCACACCACCGCATCATGTCTCATTAATGGTAGGTT               TCCAGCAGAGGTGTGGTGAATG              E. coli  O157 Lipopolysaccharide (LPS)
 
         [0000]                        E-5F           ATCCGTCACACCTGCTCTGGTGGAATGGACTAAGCTAGCTAGCGTTTTAA               AAGGTGGTGTTGGCTCCCGTAT               E-11F       ATCCGTCACACCTGCTCTGTAAGGGGGGGGAATCGCTTTCGTCTTAAGAT               GACATGGTGTTGGCTCCCGTAT               E-12F       ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGTTGG               CTCCCGTAT(59)               E-16F       ATCCGTCACACCTGCTCTATCCGTCACGCCTGCTCTATCCGTCACACCTG               CTCTGGTGTTGGCTCCCGTAT               E-17F       ATCCGTCACACCTGCTCTATCAAATGTGCAGATATCAAGACGATTTGTAC               AAGATGGTGTTGGCTCCCGTAT               E-18F       ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACGAT               AGAATGGTGTTGGCTCCCGTAT               E-19F       ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACGAT               AGAATGGTGTTGGCTCCCGTAT               E-5R       ATACGGGAGCCAACACCACCTTTTAAAACGCTAGCTAGCTTAGTCCATTC               CACCAGAGCAGGTGTGACGGAT               E-11R       ATACGGGAGCCAACACCATGTCATCTTAAGACGAAAGCGATTCCCCCCCC               TTACAGAGCAGGTGTGACGGAT               E-12R       ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAGGT               GTGACGGAT               E-16R       ATACGGGAGCCAACACCAGAGCAGGTGTGACGGATAGAGCAGGCGTGACG               GATAGAGCAGGTGTGACGGAT               E-17R       ATACGGGAGCCAACACCATCTTGTACAAATCGTCTTGATATCTGCACATT               TGATAGAGCAGGTGTGACGGAT               E-18R       ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCCAT               CTACAGAGCAGGTGTGACGGAT               E-19R       ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCCAT               CTACAGAGCAGGTGTGACGGAT            
Listeriolysin (a Surface Protein on  Listeria monocytogenes )
 
         [0000]    
       
         
               
               
             
           
               
                 LO-10F 
                   
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGTTGG 
               
               
                   
               
               
                 CTCCCGTAT 
               
               
                   
               
               
                 LO-11F 
               
               
                 ATCCGTCACACCTGCTCTGGTGGAATGGACTAAGCTAGCTAGCGTTTTAA 
               
               
                   
               
               
                 AAGGTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-13F 
               
               
                 ATCCGTCACACCTGCTCTTAAAGTAGAGGCTGTTCTCCAGACGTCGCAGG 
               
               
                   
               
               
                 AGGATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-15F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACGAT 
               
               
                   
               
               
                 AGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-16F 
               
               
                 ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACGAT 
               
               
                   
               
               
                 AGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-17F 
               
               
                 ATACGGGAGCCAACACCA 
               
               
                   
               
               
                 CAGCTGATATTGGATGGTCCGGCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-19F 
               
               
                 ATCCGTCACACCTGCTCTTGGGCAGGAGCGAGAGACTCTAATGGTAAGCA 
               
               
                   
               
               
                 AGAATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-20F 
               
               
                 ATCCGTCACACCTGCTCTCCAACAAGGCGACCGACCGCATGCAGATAGCC 
               
               
                   
               
               
                 AGGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 LO-10R 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAGGT 
               
               
                   
               
               
                 GTGACGGAT 
               
               
                   
               
               
                 LO-11R 
               
               
                 ATACGGGAGCCAACACCACCTTTTAAAACGCTAGCTAGCTTAGTCCATTC 
               
               
                   
               
               
                 CACCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-13R 
               
               
                 ATACGGGAGCCAACACCATCCTCCTGCGACGTCTGGAGAACAGCCTCTAC 
               
               
                   
               
               
                 TTTAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-15R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCCAT 
               
               
                   
               
               
                 CTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-16R 
               
               
                 ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCCAT 
               
               
                   
               
               
                 CTACAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-17R 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGTTGG 
               
               
                   
               
               
                 CTCCCGTAT 
               
               
                   
               
               
                 LO-19R 
               
               
                 ATACGGGAGCCAACACCATTCTTGCTTACCATTAGAGTCTCTCGCTCCTG 
               
               
                   
               
               
                 CCCAAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 LO-20R 
               
               
                 ATACGGGAGCCAACACCAACCTGGCTATCTGCATGCGGTCGGTCGCCTTG 
               
               
                   
               
               
                 TTGGAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
       Listeriolysin (Alternate Form of  Listeria  Surface Protein Designated “Pest-Free”) 
       [0026]      
         [0000]                        LP-3F           ATCCGTCACACCTGCTCTGTAGATGGCAAGGCATAAGCGTCCGGAACGAT               AGAATGGTGTTGGCTCCCGTAT               LP-11F       ATCCGTCACACCTGCTCTAACCAAAAGGGTAGGAGACCAAGCTAGCGATT               TGGATGGTGTTGGCTCCCGTAT               LP-13F       ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCT               GTGGTGTTGGCTCCCGTAT               LP-14F       ATCCGTCACACCTGCTCTGAAGCCTAACGGAGAAGATGGCCCTACTGCCG               TAGGTGGTGTTGGCTCCCGTAT               LP-15F       ATCCGTCACACCTGCTCTACTAAACAAGGGCAAACTGTAAACACAGTAGG               GGCGTGGTGTTGG               CTCCCGTAT               LP-17F       ATCCGTCACACCTGCTCTGGTGTTGGCTCCCGTATAGCTTGGCTCCCGTA               TGGTGTTGGCTCCCGTAT               LP-18F       ATCCGTCACACCTGCTCTGTCGCGATGATGAGCAGCAGCGCAGGAGGGAG               GGGGTGGTGTTGGCTCCCGTAT               LP-20F       ATCCGTCACACCTGCTCTGATCAGGGAAGACGCCAACACTGGTGTTGGCT               CCCGTAT               LP-3R       ATACGGGAGCCAACACCATTCTATCGTTCCGGACGCTTATGCCTTGCCAT               CTACAGAGCAGGTGTGACGGAT               LP-11R       ATACGGGAGCCAACACCATCCAAATCGCTAGCTTGGTCTCCTACCCTTTT               GGTTAGAGCAGGTGTGACGGAT               LP-13R       ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAGGT               GTGACGGAT               LP-14R       ATACGGGAGCCAACACCACCTACGGCAGTAGGGCCATCTTCTCCGTTAGG               CTTCAGAGCAGGTGTGACGGAT               LP-15R       ATACGGGAGCCAACACCACGCCCCTACTGTGTTTACAGTTTGCCCTTGTT               TAGTAGAGCAGGTGTGACGGAT               LP-17R       ATACGGGAGCCAACACCATACGGGAGCCAAGCTATACGGGAGCCAACACC               AGAGCAGGTGTGACGGAT               LP-18R       ATACGGGAGCCAACACCACCCCCTCCCTCCTGCGCTGCTGCTCATCATCG               CGACAGAGCAGGTGTGACGGAT               LP-20R       ATACGGGAGCCAACACCAGTGTTGGCGTCTTCCCTGATCAGAGCAGGTGT               GACGGAT              Salmonella typhimurium  Lipopolysaccharide (LPS)
 
         [0000]    
       
         
               
               
             
           
               
                 St-7F 
                   
               
               
                 ATCCGTCACACCTGCTCTGTCCAAAGGCTACGCGTTAACGTGGTGTTGGC 
               
               
                   
               
               
                 TCCCGTAT 
               
               
                   
               
               
                 St-10F 
               
               
                 ATCCGTCACACCTGCTCTGGAGCAATATGGTGGAGAAACGTGGTGTTGGC 
               
               
                   
               
               
                 TCCCGTAT 
               
               
                   
               
               
                 St-11F 
               
               
                 ATCCGTCACACCTGCTCTGCCGGACCATCCAATATCAGCTGTGGTGTTGG 
               
               
                   
               
               
                 CTCCCGTAT 
               
               
                   
               
               
                 St-15F 
               
               
                 ATCCGTCACACCTGCTCTGAACAGGATAGGGATTAGCGAGTCAACTAAGC 
               
               
                   
               
               
                 AGCATGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-16F 
               
               
                 ATCCGTCACACCTGCTCTGGCGGACAGGAAATAAGAATGAACGCAAAATT 
               
               
                   
               
               
                 TATCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-18F 
               
               
                 ATCCGTCACACCTGCTCTACGCAACGCGACAGGAACATTCATTATAGAAT 
               
               
                   
               
               
                 GTGTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-19F 
               
               
                 ATCCGTCACACCTGCTCTCGGCTGCAATGCGGGAGAGTAGGGGGGAACCA 
               
               
                   
               
               
                 AACCTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-20F 
               
               
                 ATCCGTCACACCTGCTCTATGACTGGAACACGGGTATCGATGATTAGATG 
               
               
                   
               
               
                 TCCTTGGTGTTGGCTCCCGTAT 
               
               
                   
               
               
                 St-7R 
               
               
                 ATACGGGAGCCAACACCACGTTAACGCGTAGCCTTTGGACAGAGCAGGTG 
               
               
                   
               
               
                 TGACGGAT 
               
               
                   
               
               
                 St-10R 
               
               
                 ATACGGGAGCCAACACCACGTTTCTCCACCATATTGCTCCAGAGCAGGTG 
               
               
                   
               
               
                 TGACGGAT 
               
               
                   
               
               
                 St-11R 
               
               
                 ATACGGGAGCCAACACCACAGCTGATATTGGATGGTCCGGCAGAGCAGGT 
               
               
                   
               
               
                 GTGACGGAT 
               
               
                   
               
               
                 St-15R 
               
               
                 ATACGGGAGCCAACACCATGCTGCTTAGTTGACTCGCTAATCCCTATCCT 
               
               
                   
               
               
                 GTTCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-16R 
               
               
                 ATACGGGAGCCAACACCAGATAAATTTTGCGTTCATTCTTATTTCCTGTC 
               
               
                   
               
               
                 CGCCAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-18R 
               
               
                 ATACGGGAGCCAACACCAACACATTCTATAATGAATGTTCCTGTCGCGTT 
               
               
                   
               
               
                 GCGTAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-19R 
               
               
                 ATACGGGAGCCAACACCAGGTTTGGTTCCCCCCTACTCTCCCGCATTGCA 
               
               
                   
               
               
                 GCCGAGAGCAGGTGTGACGGAT 
               
               
                   
               
               
                 St-20R 
               
               
                 ATACGGGAGCCAACACCAAGGACATCTAATCATCGATACCCGTGTTCCAG 
               
               
                   
               
               
                 TCATAGAGCAGGTGTGACGGAT 
               
             
          
         
       
     
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0027]      FIG. 1 . is a schematic illustration that illustrates a comparison of possible nucleic acid FRET assay formats. 
           [0028]      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. 
           [0029]      FIGS. 3A . and  3 B. are “lights on” competitive FRET-aptamer spectra and a line graph for  E. coli  bacteria using aptamers generated against various components of lipopolysaccharide (LPS) such as the rough core (Ra) antigen and the 2-keto-3-deoxyoctanate (KDO) antigen. 
           [0030]      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. 
           [0031]      FIGS. 5A . and  5 B. are “lights off” competitive FRET-aptamer spectra and line graphs in response to increasing amounts of a foot-and-mouth disease (FMD) aphthovirus surface peptide. 
           [0032]      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). 
           [0033]      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 
       [0034]    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. 
         [0035]    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. 
         [0036]    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. 
         [0037]    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. 
         [0038]    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. 
         [0039]    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 (&lt;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. 
         [0040]    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. 
         [0041]    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. 
         [0042]    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. 
         [0043]    The final FRET aptamers are able to act as one-step “lights on” or “lights off” binding and detection components in assays. 
         [0044]    Intrachain FRET-aptamers that are to be used in assays with long shelf-lives may be lyophilized (freeze-dried) and reconstituted. 
         [0045]      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. 
         [0046]      FIGS. 3A . and  3 B. are FRET fluorescence spectra and line graphs generated as a function of live  E. coli  (Crooks strain, ATCC No. 8739) concentration using LPS component competitive FRET-aptamers. Error bars represent the standard deviations of the mean for four measurements. 
         [0047]      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. 
         [0048]      FIGS. 5A . and  5 B. are FRET fluorescence spectra and line graphs generated as a function of Foot-and-Mouth Disease (FMD) peptide concentration using FMD peptide competitive FRET-aptamers. Error bars represent the standard deviations of the mean for four measurements. 
         [0049]      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. 
         [0050]      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 ) 
       [0051]    In this example, surface proteins from heat-killed  Leishmania donovani  were extracted with 3 M MgCl 2  overnight at 4° C. These proteins were then linked to tosyl-magnetic microbeads and used in a standard SELEX aptamer generation protocol. After 5 rounds of SELEX, the aptamer population was “doped” during the standard PCR reaction with 3 uM fluorescein-dUTP and purified on 10 kD molecular weight cut off spin columns. Some of the  L. donovani  surface proteins were then labeled with dabcyl-NHS ester and purified on a PD-10 (Sephadex G25) column. The dabcyl-labeled surface proteins were combined with the fluorescein-labeled aptamer population so as to produce a 1:1 fluorescein-aptamer:dabcyl-protein ratio. Thereafter, unlabeled  L. donovani  surface proteins were introduced into the assay system to compete with the labeled proteins for binding to the aptamers, thereby producing the “lights off” FRET assay results depicted in  FIGS. 2A and 2B  (fresh assay results, solid line). The assays were also examined following lyophilization (freeze drying) and reconstitution (rehydration) in the presence or absence of 10% fetal bovine serum (FBS) as a possible preservative with the results shown in  FIGS. 2A and 2B . The DNA sequences of several of these candidate  Leishmania  aptamers are given in SEQ IDs XX-XX. 
       Example 2 
     Competitive Fret-Aptamer Assay for  E. Coli  in Environmental Water Samples or Foods Using LPS Component Aptamers 
       [0052]      E. coli , especially the enterohemorrhagic strains such as O157:H7 which produce Verotoxin or Shiga toxins, are of concern in environmental water samples and foods. Their rapid detection (within minutes) with ultrasensitivity is important in protecting swimmers as well as those consuming water and foods. In this example, aptamers were generated against whole LPS from  E. coli  O111:B4 and its components such as glucosamine, KDO, and the rough mutant core antigen (Ra; lacking the outer oligosaccharide chains). In the case of glucosamine, the free primary amine in its structure enabled conjugation to tosyl-magnetic beads. KDO antigen was immobilized onto amine-conjugated magnetic beads via its carboxyl group and the bifunctional linker EDC. The rough Ra core antigen and whole LPS were linked to amine-magnetic beads via reductive amination using sodium periodate to oxidize the saccharides to aldehydes followed by the use of sodium cyanoborohydride for reductive amination as will be clear to anyone skilled in the art. Once immobilized the target-magnetic beads were used for aptamer affinity selection from a random library of 72 base aptamers (randomized 36mer flanked by known 18mer primer regions). After 5 rounds of aptamer selection and amplification, the various LPS component aptamer populations were subjected to 10 rounds of PCR in the presence of Alexa Fluor (AF) 546-14-dUTP (Invitrogen), then heated to 95° C. for 5 minutes and added to heat-killed  E. coli  O157:H7 (Kirkegaard Perry Laboraties, Inc., Gaithersburg, Md.) and used in competitive FRET-aptamer assays with various concentrations of unlabeled live  E. coli  (Crooks strain, ATCC No. 8739) resulting in the FRET spectra and line graphs shown in  FIGS. 3A and 3B . Candidate DNA aptamer sequences for detection of LPS 0111 and LPS components or associated  E. coli  and other Gram negative bacteria are given in SEQ ID Nos. XX-XX. 
       Example 3 
     Competitive FRET-Aptamer Assay for Enterococci in Environmental Water Samples 
       [0053]    Gram positive enterococci, such as  Enterococcus faecalis , are also indicators of fecal contamination of environmental water, recreational waters, or treated wastewater (effluent from sewage treatment plants). Water testers desire to detect the presence of these bacteria rapidly (within minutes) and with great sensitivity. In this example, aptamers were generated against whole lipoteichoic acid (TA; teichoic acid). TA from  E. faecalis  was immobilized on magnetic beads by reductive amination using sodium periodate to first oxidize saccharides into aldehydes followed by reductive amination using amine-magnetic beads and sodium cyanoborohydride as will be known to anyone skilled in the art. Once immobilized the target-magnetic beads were used for aptamer affinity selection from a random library of 72 base aptamers (randomized 36mer flanked by known 18mer primer regions). After 5 rounds of aptamer selection and amplification, the TA aptamer population was subjected to 10 rounds of PCR in the presence of AF 546-14-dUTP (Invitrogen), then heated to 95° C. for 5 minutes and added to live  E. faecalis . The complexes were purified by centrifugation and washing and used in competitive FRET-aptamer assays with various concentrations of unlabeled live  E. faecalis  resulting in the FRET spectra and bar graphs shown in  FIGS. 4A . and  4 B. Candidate DNA aptamer sequences for detection of lipoteichoic acid (TA) and associated enterococi or other Gram positive bacteria are given in SEQ ID Nos. XX-XX. 
       Example 4 
     Detection of Foot-And-Mouth (FMD) Disease or Other Highly Communicable Viruses Among Animal or Human Populations 
       [0054]    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 200 through 213 of 16 different O-type FMD viruses and represents a neutralizable antigenic region wherein antibodies are known to bind. The FMD peptide was immobilized on tosyl-magnetic beads via the three lysine residues in its structure. Once immobilized the target-magnetic beads were used for aptamer affinity selection from a random library of 72 base aptamers (randomized 36mer flanked by known 18mer primer regions). After 5 rounds of aptamer selection and amplification, the FMD (peptide) aptamer populations were subjected to 10 rounds of PCR in the presence of Alexa Fluor (AF) 546-14-dUTP (Invitrogen), then heated to 95° C. for 5 minutes and added to their BHQ-2-labeled-peptide target. The complexes were purified by size-exclusion chromatography over Sephadex G25 and used in competitive FRET-aptamer assays with various concentrations of unlabeled FMD peptide resulting in the FRET spectra and line graphs shown in  FIGS. 5A and 5B . Candidate DNA aptamer sequences for detection of the FMD peptide and associated strains of FMD virus are given in SEQ ID Nos. XX-XX. 
       Example 5 
     Detection of Organophosphorus (OP) Nerve Agent, Pesticides, and Acetylcholine (ACh) 
       [0055]    The use of OP nerve agents on Iraqi Kurds in the late 1980&#39;s followed by the 1995 use of sarin in a Japanese subway underscore the need for rapid and sensitive detection of OP nerve agents such as FRET-aptamer assays might provide. In addition, there is a desire in the agricultural industry to detect pesticides (also OP nerve agents) on the surfaces of fruits and vegetables in the field or in grocery stores. Finally, aptamers that bind and detect acetylcholine (ACh) may be of value in determining the impact of OP nerve agents on acetylcholinesterase (AChE) activity. Candidate aptamer sequences for the nerve agent soman, methylphosphonic acid (MPA, a common nerve agent hydrolysis product), the pesticides diazinon and malathion, and ACh are given in SEQ ID Nos. XX-XX. Amino-MPA and para-aminophenyl-soman were immobilized on tosyl-magnetic beads and used for aptamer 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. 
         [0056]    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. XX-XX), which communicate between many Gram negative bacteria such as Pseudomonads to signal proliferation and the induction of virulence factors, thereby leading to disease.
 
2) Detection and quantitation of botulinum toxins (BoNTs) for determination of the presence of biological warfare or bioterrorism agents (SEQ ID Nos. XX-XX) and  Clostridium botulinum  in vivo.
 
3) Detection and quantitation of  Campylobacter jejuni  and related  Campylobacter  species (SEQ ID Nos. XX-XX) in foods and water to prevent foodborne or waterborne illness outbreaks (add 2006 JCLA paper reference here).
 
4) Detection and quantitation of poly-D-glutamic acid (PDGA; SEQ ID Nos. XX-XX) from vegetative forms of pathogenic  Bacillus anthracis  or other similar encapsulated bacteria in vivo or in the environment to rapidly diagnose biological warfare or bioterrorist activity and provide intervention.
 
5) Detection and quantitation of  Bacillus thuringiensis  bacterial endospores in the environment to assist in biological warfare or bioterrorism detection field trials or forensic work.
 
         [0057]    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.

Technology Classification (CPC): 2