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US 11,453,646 B2Granted
Methods for RNA detection and quantification
AssigneeCornell University
InventorsSamie R. Jaffrey, Jeremy Paige
Life of this patent
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Abstract
The present invention relates to novel nucleic acid molecules, called aptamers, that bind specifically to a small molecule fluorophore and thereby enhance the fluorescence signal of the fluorophore upon exposure to radiation of suitable wavelength. Molecular complexes formed between the novel fluorophores, novel nucleic acid molecules, and their target molecules are described, and the use of multivalent aptamer constructs as fluorescent sensors for target molecules of interest are also described.
Claims14
A nucleic acid molecule that binds to a compound according to formula I below:
- wherein,
- Q is S or O;
- Y is N;
- Z is N or C(R10);
- Ar is phenyl;
- R1 is —(CH2)n—R6 where n is an integer greater than or equal to 1;
- R2 is methyl, mono-halo methyl, di-halo methyl, tri-halo methyl, aldoxime, O-methyl-aldoxime, iminomethyl, carboxylic acid, thioic acid, (thio)amido, alkyl(thio)amido, unsubstituted or substituted phenyl with up to three substituents (R7-R9), (meth)acrylate, or C2-8 unsaturated hydrocarbon optionally terminated with amine, amide, carboxylic acid, ester, enone, oxime, O-methyl-oxime, imine, nitromethane, nitrile, ketone, mono-halo, di-halo, tri-halo, nitro, cyano, acrylonitrile, acrylonitrile-enoate, acrylonitrile-carboxylate, acrylonitrile-amide, alkylester, or a second aromatic or hetero-aromatic ring;
- R3-R5 are independently selected from H, hydroxy, alkyl, alkoxy, fluoro, chloro, bromo, mono-halo alkoxy, di-halo alkoxy, tri-halo alkoxy, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, thioc acid, alkylester, a surface-reactive group, a solid surface, and a functional group that can be linked to a reactive group on the solid surface;
- R6 is hydroxy, alkoxy, fluoro, chloro, bromo, mono-halo methyl, di-halo methyl, tri-halo alkoxy, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, or alkylester;
- and
- R7-R10 are independently selected from H, hydroxy, alkyl, alkoxy, fluoro, chloro, bromo, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, thioic acid, and alkylester, wherein binding of the nucleic acid molecule to the compound of formula (I) induces fluorescence of said compound.
8 more elements
A molecular complex comprising:
- a compound according to formula I below:
- wherein,
- Q is S or O;
- Y is N;
- Z is N or C(R10);
- Ar is phenyl;
- R1 is —(CH2)n—R6 where n is an integer greater than or equal to 1;
- R2 is methyl, mono-halo methyl, di-halo methyl, tri-halo methyl, aldoxime, O-methyl-aldoxime, iminomethyl, carboxylic acid, thioic acid, (thio)amido, alkyl(thio)amido, unsubstituted or substituted phenyl with up to three substituents (R7-R9), (meth)acrylate, or C2-8, unsaturated hydrocarbon, wherein said C2-8 unsaturated hydrocarbon is optionally terminated with amine, amide, carboxylic acid, ester, enone, oxime, O-methyl-oxime, imine, nitromethane, nitrile, ketone, mono-halo, di-halo, tri-halo, nitro, cyano, acrylonitrile, acrylonitrile-enoate, acrylonitrile-carboxylate, acrylonitrile-amide, alkylester, or a second aromatic or hetero-aromatic ring;
- R3-R5 are independently selected from H, hydroxy, alkyl, alkoxy, fluoro, chloro, bromo, mono-halo alkoxy, di-halo alkoxy, tri-halo alkoxy, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, thioc acid, alkylester, a surface-reactive group, a solid surface, and a functional group that can be linked to a reactive group on the solid surface;
- R6 is hydroxyl, alkoxy, fluoro, chloro, bromo, mono-halo alkoxy, di-halo alkoxy, tri-halo alkoxy, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, alkylester;
- and
- R7-R10 are independently selected from H, hydroxy, alkyl, alkoxy, fluoro, chloro, bromo, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, and mono-, di-, or tri-halo methyl, ketone, carboxylic acid, thioic acid, and alkylester;
- and
- a nucleic acid molecule bound specifically to the compound,
- wherein, upon specific binding of the nucleic acid molecule to the compound of formula (I) and upon exposure to radiation of suitable wavelength, the compound exhibits an increase in quantum yield in comparison to the quantum yield of the compound prior to specific binding.
12 more elements
A kit comprising:
- a compound according to formula I below:
- wherein,
- O is S or O;
- Y is N;
- Z is N or C(R10);
- Ar is phenyl;
- R1 is —(CH2)n—R6 where n is an integer greater than or equal to 1;
- R2 is methyl, mono-halo methyl, di-halo methyl, tri-halo methyl, aldoxime, an O-methyl-aldoxime, iminomethyl, carboxylic acid, thioic acid, (thio)amido, alkyl(thio)amido, unsubstituted or substituted phenyl with up to three substituents (R7-R9), (meth)acrylate, or C2-8 unsaturated hydrocarbon, wherein said C2-8 unsaturated hydrocarbon is optionally terminated with amine, amide, carboxylic acid, ester, enone, oxime, O-methyl-oxime, imine, nitromethane, nitrile, ketone, mono-halo, di-halo, tri-halo, nitro, cyano, acrylonitrile, acrylonitrile-enoate, acrylonitrile-carboxylate, acrylonitrile-amide, alkylester, or a second aromatic or hetero-aromatic ring;
- R3-R5 are independently selected from H, hydroxy, alkyl, alkoxy, fluoro, chloro, bromo, mono-halo alkoxy, di-halo alkoxy, tri-halo alkoxy, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, thioc acid, alkylester, a surface-reactive group, a solid surface, and a functional group that can be linked to a reactive group on the solid surface;
- R6 is hydroxy, alkoxy, fluoro, chloro, bromo, mono-halo alkoxy, di-halo alkoxy, tri-halo alkoxy, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, or alkylester;
- and
- R7-R10 are independently selected from H, hydroxy, alkyl, alkoxy, fluoro, chloro, bromo, amino, alkylamino, dialkylamino, (thio)amido, alkyl(thio)amido, alkylthio, cyano, mercapto, nitro, mono-halo methyl, di-halo methyl, tri-halo methyl, ketone, carboxylic acid, thioic acid, and alkylester;
- and
- a nucleic acid molecule that binds to the compound of formula (I) to induce fluorescence thereof,
- wherein, upon specific binding of the nucleic acid molecule to the compound of formula (I) and upon exposure to radiation of suitable wavelength, the compound exhibits an increase in quantum yield in comparison to the quantum yield of the compound prior to specific binding.
12 more elements
Dependencies are inferred from the claim text.