Bioluminescent detection of DNA synthesis
AssigneePromega Corporation
InventorsWenhui Zhou, Hui Wang, Min Zhou, Natasha Karassina, James Cali, Jolanta Vidugiriene
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Abstract
Provided herein are reagents and methods for incorporating modified nucleotides into DNA and detecting DNA synthesis. In particular, haloalkyl-modified nucleobases are provided for incorporation into nucleic acids for detection by bioluminescent binding agents.
Claims14
A compound of formula (I):
- or a salt thereof, wherein:
- B is a nucleobase selected from adenine, guanine, uracil, and cytosine;
- L is a linker selected from:
- wherein p, q, r, and s are each independently 1-6;
- and
- A is a haloalkyl group.
A nucleic acid produced by incorporating haloalkyl group modified nucleosides into DNA replication within a cell, the method comprising contacting the cell with the compound of formula (I):
- or a salt thereof, wherein:
- B is a nucleobase selected from adenine, guanine, uracil, and cytosine;
- L is a linker selected from:
- wherein p, q, r, and s are each independently 1-6;
- and
- A is a haloalkyl group;
- allowing the compound to enter the cell;
- allowing the compound to be converted into a modified deoxynucleotide triphosphate by enzymes within a cell;
- and
- further allowing the modified deoxynucleotide triphosphate to be incorporated into DNA replication during DNA synthesis.
A method of detecting haloalkyl-labelled nucleic acid within a cell comprising:
- (a) lysing the cell to produce a cell lysate comprising the haloalkyl-labelled nucleic acid;
- (b) contacting the cell lysate with (i) a first fusion of (A) a modified dehalogenase capable of covalently binding the haloalkyl group and (B) a first component of a reporter complex, and (ii) a second fusion of (A) a modified dehalogenase capable of covalently binding the haloalkyl group and (B) a second component of a reporter complex, wherein the detectable complex is capable of producing a detectable signal upon the first component being brought in contact or physical proximity with the second component;
- (c) allowing the modified dehalogenase to bind to the haloalkyl groups on the nucleic acid;
- and
- (d) detecting the detectable signal from the reporter complex.
A method of detecting haloalkyl-labelled nucleic acid within a cell comprising:
- (a) lysing the cell to produce a cell lysate comprising the haloalkyl-labelled nucleic acid;
- (b) contacting the cell lysate with (i) (A) a modified dehalogenase capable of covalently binding the haloalkyl group tethered to (B) a reporter capable of emitting light at a first wavelength, and (ii) (A) a modified dehalogenase capable of covalently binding the haloalkyl group and (B) fluorophore with an excitation spectrum that overlaps the first wavelength and an emission spectrum;
- (c) allowing the modified dehalogenase to bind to the haloalkyl groups on the nucleic acid;
- and
- (d) detecting a wavelength within the emission spectrum of the fluorophore.
Dependencies are inferred from the claim text.