Compounds and methods for fluorescently labeling nucleic acids
OwnerJonathan H Davisinventor
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Abstract
The invention provides methods for the selection of nucleic acids that bind and thereby modulate (i.e, increase or decrease) the fluorescence intensity of a fluorophore. These selected nucleic acids may be used in methods, for example, for the detection, visualization, or quantitation of nucleic acids of interest.
Claims47
A method of selecting an RNA molecule which binds a fluorophore, wherein said binding increases the fluorescence intensity of said fluorophore, said method comprising the steps of:
- (a) providing a population of candidate RNA molecules;
- (b) selecting said candidate RNA molecules which bind said fluorophore;
- (c) contacting said candidate RNA molecules which bind said fluorophore with said fluorophore;
- and
- (d) selecting said RNA molecules which, upon binding said fluorophore, increase its fluorescence intensity.
A method of selecting a DNA molecule which binds a fluorophore, wherein said binding increases the fluorescence intensity of said fluorophore, said method comprising the steps of:
- (a) providing a population of candidate DNA molecules;
- (b) selecting said candidate DNA molecules which bind said fluorophore;
- (c) contacting said candidate DNA molecules which bind said fluorophore with said fluorophore;
- and
- (d) selecting said DNA molecules which, upon binding said fluorophore, increase its fluorescence intensity.
A method of determining the presence, location, or quantity of an RNA of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said RNA of interest covalently linked to an RNA aptamer;
- (b) contacting said cell or said sample with a fluorophore, whereby said aptamer binds to said fluorophore and increases its fluorescence intensity;
- and
- (c) visualizing or measuring the fluorescence of said fluorophore, thereby determining the presence, location, or quantity of said RNA of interest in said cell or said in vitro sample.
A method of determining the presence, location, or quantity of a DNA of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion DNA comprising said DNA of interest covalently linked to a DNA aptamer;
- (b) contacting said cell or said sample with a fluorophore, whereby said aptamer binds to said fluorophore and increases its fluorescence intensity;
- and
- (c) visualizing or measuring the fluorescence of said fluorophore, thereby determining the presence, location, or quantity of said DNA of interest in said cell or said in vitro sample.
A method of determining whether a compound is capable of modulating the transcription of an RNA of interest, said method comprising the steps of:
- (a) expressing in a cell or an in vitro sample a fusion RNA comprising said RNA of interest covalently linked to an RNA aptamer;
- (b) contacting said cell or said sample with either said compound and said fluorophore or with said fluorophore alone, whereby said aptamer binds to said fluorophore and increases its fluorescence intensity;
- and
- (c) measuring said fluorescence intensity in the presence and absence of said compound, whereby said compound is determined to modulate said transcription if said compound effects a change in said fluorescence intensity.
A method of determining whether a compound modulates the half-life of an RNA of interest, said method comprising the steps of:
- (a) expressing in a cell or an in vitro sample a fusion RNA comprising said RNA of interest covalently linked to an RNA aptamer,
- (b) contacting said cell or said sample with either said compound and a fluorophore or with a fluorophore alone, whereby said RNA aptamer binds to said fluorophore and increases its fluorescence intensity;
- and
- (c) measuring said fluorescence intensity in the presence and absence of said compound, whereby said compound is determined to modulate said half-life if said compound effects a change in said fluorescence intensity.
A method of determining whether an antisense nucleic acid binds a target RNA in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said target RNA covalently linked to an RNA aptamer;
- (b) contacting said cell or said sample with either said antisense nucleic acid and a fluorophore or with a fluorophore alone, whereby said RNA aptamer binds to said fluorophore and increases its fluorescence intensity;
- and
- (c) measuring said fluorescence intensity in the presence and absence of said antisense nucleic acid, whereby said antisense nucleic acid is determined to bind said target RNA if said antisense nucleic acid decreases said fluorescence intensity.
A method of determining whether an antisense nucleic acid binds a target RNA in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said target RNA covalently linked to an RNA aptamer which binds a first fluorophore;
- (b) contacting said cell or said sample with either said first fluorophore and said antisense nucleic acid covalently linked to a second fluorophore or with said first fluorophore alone, whereby said RNA aptamer binds to said first fluorophore and increases its fluorescence intensity;
- and wherein the emission wavelength of said first fluorophore is different from that of said second fluorophore, and the emission wavelength of said second fluorophore induces the fluorescence of said first fluorophore;
- and
- (c) measuring said fluorescence intensity of said first fluorophore in the presence and absence of said antisense nucleic acid;
- whereby said antisense nucleic acid is determined to bind said target RNA if said antisense nucleic acid increases said fluorescence -intensity of said first fluorophore.
A method of determining whether an antisense nucleic acid binds a target RNA in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said target RNA covalently linked to an RNA aptamer which binds a fluorophore;
- (b) contacting said cell or said sample with either said fluorophore and said antisense nucleic acid covalently linked to a quencher or with said fluorophore alone, whereby said RNA aptamer binds to said fluorophore and increases its fluorescence intensity;
- and wherein the fluorescence intensity of said fluorophore is decreased by said quencher;
- and
- (e) measuring said fluorescence intensity of said fluorophore in the presence and absence of said antisense nucleic acid;
- whereby said antisense nucleic acid is determined to bind said target RNA if said antisense nucleic acid decreases said fluorescence intensity.
A method of determining whether an antisense nucleic acid binds a target RNA in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said target RNA covalently linked to an RNA aptamer which binds a first fluorophore;
- (b) contacting said cell or said sample with either said first fluorophore, said antisense nucleic acid covalently linked to a nucleic acid aptamer which binds a second fluorophore, and said second fluorophore or with said first fluorophore alone, whereby said RNA aptamer binds to said first fluorophore and increases its fluorescence intensity, and said nucleic acid aptamer binds to said second fluorophore and increases its fluorescence intensity;
- and wherein the emission wavelength of said first fluorophore is different from that of said second fluorophore, and the emission wavelength of said second fluorophore induces the fluorescence of said first fluorophore;
- and
- (c) measuring said fluorescence intensity of said first fluorophore in the presence and absence of said antisense nucleic acid, whereby said antisense nucleic acid is determined to bind said target RNA if said antisense nucleic acid increases said fluorescence intensity of first fluorophore.
A method of determining whether an antisense nucleic acid binds a target RNA in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said target RNA covalently linked to an RNA aptamer which binds a fluorophore;
- (b) contacting said cell or said sample with said fluorophore, said antisense nucleic acid covalently linked to a nucleic acid aptamer which binds a quencher, and said quencher, or with said fluorophore alone, whereby said RNA aptamer binds to said fluorophore and increases its fluorescence intensity, and said nucleic acid aptamer binds to said quencher;
- and wherein the fluorescence intensity of said fluorophore is decreased by said quencher;
- and
- (c) measuring said fluorescence intensity of said fluorophore in the presence and absence of said antisense nucleic acid, whereby said antisense nucleic acid is determined to bind said target RNA if said antisense nucleic acid decreases said fluorescence intensity.
A method of determining whether a first RNA is co-localized with a second RNA of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a first fusion RNA comprising said first RNA covalently linked to a first RNA aptamer which binds a first fluorophore;
- (b) expressing in said cell or said sample a second fusion RNA comprising said second RNA of interest covalently linked to a second RNA aptamer which binds a second fluorophore, wherein the emission wavelength of said second fluorophore is different from that of said first fluorophore;
- (c) contacting said cell or said sample with said first fluorophore and said second fluorophore, whereby said first fluorophore binds to said first RNA aptamer and said second fluorophore binds to said second RNA aptamer, and whereby said binding increases the fluorescence intensity of said first fluorophore and said second fluorophore;
- and
- (d) visualizing the fluorescence of said first fluorophore and said second fluorophore, whereby said first RNA of interest is determined to co-localize with said second RNA of interest if said fluorescence of said first fluorophore is detected proximal to the fluorescence of said second fluorophore.
A method of determining whether an RNA of interest is co-localized with a protein of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said RNA of interest covalently linked to an RNA aptamer which binds a first fluorophore;
- (b) expressing in said cell or said sample a fusion protein comprising said protein of interest covalently linked to a detectable protein;
- (c) contacting said cell or said sample with said first fluorophore, whereby said RNA aptamer binds to said first fluorophore and increases its fluorescence intensity;
- and
- (d) visualizing the fluorescence of said first fluorophore and determining the localization of said detectable protein, whereby said first RNA of interest is determined to co-localize with said protein of interest if said fluorescence of said first fluorophore is detected proximal to said detectable protein.
A method of determining whether a first RNA of interest interacts with a second RNA of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a first fusion RNA comprising said first RNA of interest covalently linked to a first RNA aptamer which binds a first fluorophore;
- (b) expressing in said cell or said sample a second fusion RNA comprising said second RNA of interest covalently linked to a second RNA aptamer which binds a second fluorophore, wherein the emission wavelength of said first fluorophore is different from that of said second fluorophore, and wherein the emission wavelength of said first fluorophore induces the fluorescence of said second fluorophore;
- (c) contacting said cell or said sample with either said first fluorophore and said second fluorophore or with said second fluorophore alone, whereby said first fluorophore binds to said first RNA aptamer and said second fluorophore binds to said second RNA aptamer, and whereby said binding increases the fluorescence intensity of said first fluorophore and said second fluorophore;
- and
- (d) measuring said fluorescence intensity of said second fluorophore in the presence and absence of said first fluorophore, whereby said first RNA of interest is determined to interact with said second RNA of interest if said first fluorophore induces said fluorescence intensity of said second fluorophore.
A method of determining whether an RNA of interest interacts with a protein of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said RNA of interest covalently linked to a RNA aptamer which binds a first fluorophore;
- (b) expressing in said cell or said sample a fusion protein comprising said protein of interest covalently linked to detectable protein which binds a second fluorophore, wherein the emission wavelength of said first fluorophore is different from that of said second fluorophore, and wherein the emission wavelength of said first fluorophore induces the fluorescence of said second fluorophore or the emission wavelength of said second fluorophore induces the fluorescence of said first fluorophore;
- (c) contacting said cell or said sample with either said first fluorophore and said second fluorophore, said first fluorophore alone, or said second fluorophore alone, whereby said RNA aptamer binds to said first fluorophore and increases its fluorescence intensity, and whereby said detectable protein binds to said second fluorophore and increases its fluorescence intensity;
- and
- (d) measuring said fluorescence intensity of said first fluorophore in the presence and absence of said second fluorophore or measuring said fluorescence intensity of said second fluorophore in the presence and absence of said first fluorophore, whereby said RNA of interest is determined to interact with said protein of interest if fluorescence resonance energy transfer occurs between said first fluorophore and said second fluorophore.
A method of determining whether an RNA of interest interacts with a protein of interest in a cell or an in vitro sample, said method comprising the steps of:
- (a) expressing in said cell or said sample a fusion RNA comprising said RNA of interest covalently linked to a RNA aptamer which binds a fluorophore;
- (b) expressing in said cell or said sample a fusion protein comprising said protein of interest covalently linked to detectable protein with intrinsic fluorescence, wherein the emission wavelength of said fluorophore is different from that of said detectable protein, and wherein the emission wavelength of said fluorophore induces the fluorescence of said detectable protein or wherein the emission wavelength of said detectable protein induces the fluorescence of said fluorophore;
- (c) contacting said cell or said sample with said fluorophore, whereby said RNA aptamer binds to said fluorophore and increases its fluorescence intensity;
- and
- (d) measuring said fluorescence intensity of said fluorophore in the presence and absence of said detectable protein or measuring said fluorescence intensity of said detectable protein in the presence and absence of said fluorophore, whereby said RNA of interest is determined to interact with said protein of interest if fluorescence resonance energy transfer occurs between said fluorophore and said detectable protein.
A population of nucleic acids having two loops separated by a base-paired helix, wherein at least one nucleic acid has a non-naturally-occurring polynucleotide sequence.
A population of nucleic acids having a loop flanked by a base-paired helix on one side and a hairpin on the other side, wherein at least one nucleic acid has a non-naturally-occurring polynucleotide sequence.
A population of nucleic acids having a pseudoknot structure, wherein at least one nucleic acid has a non-naturally-occurring polynucleotide sequence.
Dependencies are inferred from the claim text.