Methods of sequencing fluorophore-quencher FRET-aptamers

Abstract

The present invention describes methods for the production and selecting 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. Alternatively, single DNA or RNA intrachain FRET-aptamers can be sequenced and the locations of F and Q within the structure can be determined by nanopore sequencing and the locations of F and Q within the structure can be verified by nucleic acid “combing” coupled to high-powered fluorescence microscopy.

1 . A method of determining the locations of fluorophores (“F”) or quenchers (“Q”) bound to a single-chain fluorescence resonance energy transfer (“FRET”)-aptamer that selectively binds to a target molecule, comprising:
 selecting a FRET-aptamer that exhibits a measurable change in fluorescence between the fluorescence of said FRET-aptamer when it is not bound to said target molecule compared to the fluorescence of said FRET-aptamer when it is bound to said target molecule; and 
 determining the locations of fluorophores (“F”) or quenchers (“Q”) of said FRET-aptamer via single FRET-aptamer sequencing. 
 
     
     
         2 . The method of  claim 1 , wherein said single FRET-aptamer sequencing comprises nanopore sequencing. 
     
     
         3 . The method of  claim 2 , wherein said nanopore sequencing further comprises:
 drawing an individual single-stranded DNA or RNA molecule through a pore wherein said pore is greater than or equal to 1 nm in diameter;   measuring changes in optical properties as each DNA or RNA base or nucleotide, fluorophore (“F”), or quencher (“Q”) moves through said pore; and   identifying said DNA bases, Fs, and Q's based upon said measured optical property changes.   
     
     
         4 . The method of  claim 3 , wherein said pore is in an inorganic silicon nitride or biological phospholipid bilayer membrane. 
     
     
         5 . The method of  claim 3 , wherein said drawing step is accomplished by pulling said DNA or RNA molecule through said pore. 
     
     
         6 . The method of  claim 5 , wherein said pulling of said DNA or RNA molecule through said pore is accomplished via applying electrophoresis or negative pressure to said DNA or RNA molecule. 
     
     
         7 . The method of  claim 3 , wherein said drawing step is accomplished by pushing said DNA or RNA molecule through said pore. 
     
     
         8 . The method of  claim 7 , wherein said pushing of said DNA molecule through said pore is accomplished via applying electrophoresis or positive pressure to said DNA molecule. 
     
     
         9 . The method of  claim 2 , wherein said nanopore sequencing further comprises:
 drawing an individual single-stranded DNA molecule through a pore wherein said pore is greater than or equal to 1 nm in diameter;   measuring changes in electrical properties as each base or nucleotide moves through said pore; and   identifying said DNA or RNA bases, Fs, and Q's based upon said measured electrical property changes.   
     
     
         10 . The method of  claim 9 , wherein said pore is in an inorganic silicon nitride or biological phospholipid bilayer membrane. 
     
     
         11 . The method of  claim 9 , wherein said drawing step is accomplished by pulling said DNA molecule through said pore. 
     
     
         12 . The method of  claim 11 , wherein said pulling of said DNA or RNA molecule through said pore is accomplished via applying electrophoresis or negative pressure to said DNA or RNA molecule. 
     
     
         13 . The method of  claim 9 , wherein said drawing step is accomplished by pushing said DNA molecule through said pore. 
     
     
         14 . The method of  claim 13 , wherein said pushing of said DNA or RNA molecule through said pore is accomplished via applying electrophoresis or positive pressure to said DNA or RNA molecule. 
     
     
         15 . The method of  claim 1 , wherein said single FRET-aptamer sequencing comprises DNA or RNA combing. 
     
     
         16 . The method of  claim 15 , wherein said DNA or RNA combing further comprises:
 straightening an individual single-stranded DNA or RNA molecule;   staining said individual single-stranded DNA or RNA molecule with a nucleic acid-specific fluorescent dye;   imaging said straightened individual single-stranded DNA or RNA molecule with a fluorescence microscope;   detecting Fs along the length of said individual single-stranded DNA or RNA molecule using their emission color;   measuring a distance from either end of said individual single-stranded DNA or RNA molecule to determine a position of said F in said individual single-stranded DNA or RNA molecule.   
     
     
         17 . A method of sequencing single-chain fluorescence resonance energy transfer (“FRET”)-aptamers that contain fluorophores or quenchers in the same single-stranded oligonucleotides, comprising:
 incorporating fluorophores and quenchers into an aptamer population using polymerase chain reaction (“PCR”) wherein at least one of said fluorophores and at least one of said quenchers are incorporated into some of said oligonucleotides at random locations in said oligonucleotides; 
 exposing said aptamer population to a population of target molecules; 
 separating those FRET-aptamers that have bound to a target molecule; 
 selecting a preferred FRET-aptamer that exhibits a measurable change in fluorescence between the fluorescence of said FRET-aptamer when it is not bound to said target molecule compared to the fluorescence of said FRET-aptamer when it is bound to said target molecule; and 
 determining the nucleotide sequence of said preferred FRET-aptamer via single FRET-aptamer sequencing. 
 
     
     
         18 . The method of  claim 17 , wherein said single FRET-aptamer sequencing further comprises:
 drawing an individual single-stranded DNA or RNA molecule through a pore wherein said pore is greater than or equal to 1 nm in diameter;   measuring changes in optical properties as each DNA or RNA base, fluorophore (“F”), or quencher (“Q”) moves through said pore; and   identifying said DNA or RNA bases, Fs, and Q's based upon said measured optical property changes.   
     
     
         19 . The method of  claim 17 , wherein said single FRET-aptamer sequencing further comprises:
 drawing an individual single-stranded DNA or RNA molecule through a pore wherein said pore is greater than or equal to 1 nm in diameter;   measuring changes in optical properties as each DNA or RNA base, fluorophore (“F”), or quencher (“Q”) moves through said pore; and   identifying said DNA or RNA bases, Fs, and Q's based upon said measured electrical property changes.   
     
     
         20 . The method of  claim 17 , wherein said nanopore sequencing further comprises:
 straightening an individual single-stranded DNA or RNA molecule;   staining said individual single-stranded DNA or RNA molecule with a nucleic acid-specific fluorescent dye;   imaging said straightened individual single-stranded DNA or RNA molecule with a fluorescence microscope;   detecting Fs along the length of said individual single-stranded DNA or RNA molecule using their emission color;   measuring a distance from either end of said individual single-stranded DNA or RNA molecule to determine a position of said F in said individual single-stranded DNA or RNA molecule.