Methods of running assays using intrachain fluorophore-quencher FRET-aptamers

Abstract

The present invention describes methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis.

1 . A method of assaying a target molecule in a solution using single-stranded fluorescence resonance energy transfer (“FRET”)-aptamers, comprising:
 selecting FRET-aptamers that bind with said target molecule; 
 adding said selected FRET-aptamers to said solution, 
 wherein said FRET-aptamers have at least one fluorophore (“F”) and at least one quencher (“Q”) incorporated into said FRET-aptamers in the interior of said FRET-aptamers; 
 wherein said F and said Q are spectrally matched such that an absorption spectrum of said Q overlaps significantly with an emission spectrum of said F; 
 wherein prior to binding said target molecule said location of said F is within a Förster distance of said Q such that said Q interacts with said F such that minimal fluorescence is detectable; 
 wherein said FRET-aptamers emit increased detectable fluorescence after binding said target molecule due to an increase in said Förster distance between said Q and said F, and said detectable fluorescence changes proportionately in response to the amount of said target molecule in said solution; 
 measuring said fluorescence light level; and 
 determining the presence or absence of said target molecules in said solution. 
 
     
     
         2 . The method of  claim 1 , further comprising calculating the amount of said target molecules in said solution 
     
     
         3 . The method of  claim 2 , wherein said assay is used for detecting and quantifying a target molecule, wherein said target molecule is less than 1,000 Daltons. 
     
     
         4 . The method of  claim 3 , wherein said target molecule is selected from the group consisting of:
 pesticides, natural and synthetic amino acids, histidine, histamine, homocysteine, DOPA, melatonin, nitrotyrosine, short chain proteolysis products, cadaverine, putrescine, polyamines, spermine, spermidine, nitrogen bases of DNA or RNA, nucleosides, nucleotides, nucleotide cyclical isoforms, cAMP, cGMP, cellular metabolites, urea, uric acid, pharmaceuticals, therapeutic drugs, narcotics, hallucinogens, gamma-hydroxybutyrate, cellular mediators, cytokines, chemokines, immune modulators, neural modulators, inflammatory modulators, prostaglandins, prostaglandin metabolites, explosives, trinitrotoluene, peptides, macromolecules, proteins, bacterial surface proteins, glycoproteins, lipids, glycolipids, nucleic acids, polysaccharides, lipopolysaccharides, whole cells, and subcellular organelles or cellular fractions.   
     
     
         5 . The method of  claim 2 , wherein said assay is used for detecting and quantifying a target molecule, wherein said target molecule is equal to or greater than 1,000 Daltons and water- soluble. 
     
     
         6 . A method of assaying a target molecule in a solution using single-stranded fluorescence resonance energy transfer (“FRET”)-aptamers, comprising:
 selecting FRET-aptamers that bind with said target molecule; 
 adding said selected FRET-aptamers to said solution, 
 wherein said FRET-aptamers have at least one fluorophore (“F”) and at least one quencher (“Q”) incorporated into said FRET-aptamers in the interior of said FRET-aptamers; 
 wherein said F and said Q are spectrally matched such that an absorption spectrum of said Q overlaps significantly with an emission spectrum of said F; 
 wherein prior to binding said target molecule said location of said F is beyond a Förster distance of said Q such that said Q does not interact with said F such that said F emits detectable fluorescence 
 wherein said FRET-aptamers emit decreased detectable fluorescence after binding said target molecule due to a decrease in the Förster distance between said F and said Q, and said detectable fluorescence changes proportionately in response to the amount of said target molecule in said solution; 
 measuring said fluorescence light level; and 
 determining the presence or absence of said target molecules in said solution. 
 
     
     
         7 . The method of  claim 6 , wherein said assay is used for detecting a target molecule, wherein said target molecule is less than 1,000 Daltons. 
     
     
         8 . The method of  claim 7 , wherein said target molecule is selected from the group consisting of: pesticides, natural and synthetic amino acids, histidine, histamine, homocysteine, DOPA, melatonin, nitrotyrosine, short chain proteolysis products, cadaverine, putrescine, polyamines, spermine, spermidine, nitrogen bases of DNA or RNA, nucleosides, nucleotides, nucleotide cyclical isoforms, cAMP, cGMP, cellular metabolites, urea, uric acid, pharmaceuticals, therapeutic drugs, narcotics, hallucinogens, gamma-hydroxybutyrate, cellular mediators, cytokines, chemokines, immune modulators, neural modulators, inflammatory modulators, prostaglandins, prostaglandin metabolites, explosives, trinitrotoluene, peptides, macromolecules, proteins, bacterial surface proteins, glycoproteins, lipids, glycolipids, nucleic acids, polysaccharides, lipopolysaccharides, whole cells, and subcellular organelles or cellular fractions. 
     
     
         9 . The method of  claim 6 , wherein said assay is used for detecting a target molecule, wherein said target molecule is equal to or greater than 1,000 Daltons and water-soluble.