Method for detecting polynucleotide using RISC
Disclosed herein is a method for detecting small RNAs and discriminating single nucleotide polymorphism at a single molecule level, and specifically to a method for detection of small RNAs, whereby the detection and quantitation of small RNAs and the identification of single nucleotide polymorphism (SNP) can be achieved.
1 . A method for detecting a polynucleotide in a biological sample, the method comprising:
(a) contacting a composition for detecting the polynucleotide with the biological sample,
wherein the composition for detecting the polynucleotide comprises a first protein-nucleic acid complex comprising an Argonaute protein associated with a first nucleic acid molecule and a second protein-nucleic acid complex comprising an Argonaute protein associated with a second nucleic acid molecule,
wherein the first nucleic acid molecule comprises a nucleic acid sequence complementary to a first target nucleic acid region in the polynucleotide to be detected, and the second nucleic acid molecule comprises a nucleic acid sequence complementary to a second target nucleic acid region in the polynucleotide to be detected, wherein the first target nucleic acid region and the second target nucleic acid region differ from each other, and wherein the first nucleic acid molecule is conjugated with a first fluorophore and the second nucleic acid molecule is conjugated with a second fluorophore, wherein the first fluorophore differs from the second fluorophore,
wherein the first fluorophore generates a first fluorescent signal, and the second fluorophore generates a second fluorescent signal;
(b) detecting the first and the second fluorescent signals resulting from the contacting of (a), wherein the first fluorescent signal is a fluorescent signal generated by the first fluorophore conjugated with the first nucleic acid sequence bound to the first target nucleic acid region and the second fluorescent signal is a fluorescent signal generated by the second fluorophore conjugated with the second nucleic acid sequence bound to the second target nucleic acid region;
(c) calculating time fractions of bound state for the first fluorescent signal and for the second fluorescent signal according to formula:
Time fraction of bound state= T/P.
wherein T is a total time of sensing the first or the second fluorescent signal during a detection time, and P is the detection time; and
(d) determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than a first threshold value set for the first target nucleic acid region and the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than a second threshold value set for the second target nucleic acid region,
wherein each of the first and the second threshold values is a value in a range of from 0.03 to 0.12.
2 . The method of claim 1 , wherein the polynucleotide is selected from the group consisting of DNA, RNA, miRNA, and a combination thereof.
3 . The method of claim 1 , wherein the biological sample is an isolated cell, a cytolysate, a cell extract, a cell lysate, or an isolated DNA or RNA.
4 . The method of claim 1 , wherein the biological sample comprises a polynucleotide which has a nucleotide sequence homology of 90% or higher to the polynucleotide to be detected.
5 . The method of claim 1 , wherein the step of detecting fluorescent signals is carried out using at least one selected from the group consisting of Total Internal Reflection Fluorescence Microscopy (TIRF), confocal microscopy, Epi-fluorescence microscopy, HiLo microscopy, and Line-scanning confocal microscopy.
6 . The method of claim 1 , wherein the polynucleotide to be detected has a polynucleotide tail at an end thereof and is immobilized onto a detection chip.
7 . A method for detecting a single nucleotide polymorphism (SNP) in a polynucleotide, of the method comprising:
(a) contacting a composition for detecting the polynucleotide with a biological sample comprising the polynucleotide to be detected,
wherein the composition for detecting the polynucleotide comprises a first protein-nucleic acid complex comprising an Argonaute protein associated with a first nucleic acid molecule and a second protein-nucleic acid complex comprising an Argonaute protein associated with a second nucleic acid molecule,
wherein the first nucleic acid molecule comprises a nucleic acid sequence complementary to a first target nucleic acid region in the polynucleotide to be detected, and the second nucleic acid molecule comprises a nucleic acid sequence complementary to a second target nucleic acid region in the polynucleotide to be detected, wherein the first target nucleic acid region and the second target nucleic acid region differ from each other, and wherein the first nucleic acid molecule is conjugated with a first fluorophore, and the second nucleic acid molecule is conjugated with a second fluorophore, wherein the first fluorophore differs from the second fluorophore,
wherein the first fluorophore generates a first fluorescent signal, and the second fluorophore generates a second fluorescent signal;
(b) detecting the first and the second fluorescent signals resulting from the contacting of (a), wherein the first fluorescent signal is a fluorescent signal generated by the first fluorophore conjugated with the first nucleic acid sequence bound to the first target nucleic acid region and the second fluorescent signal is a fluorescent signal generated by the second fluorophore conjugated with the second nucleic acid sequence bound to the second target nucleic acid region;
(c) calculating time fractions of bound state for the first fluorescent signal and for the second fluorescent signal according to formula:
Time fraction of bound state= T/P.
wherein T is a total time of sensing the first or the second fluorescent signal during a detection time, and P is the detection time; and
(d) determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than a first threshold value set for the first target nucleic acid region and the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than a second threshold value set for the second target nucleic acid region,
wherein each of the first and the second threshold values is a value in a range of from 0.03 to 0.12.
8 . The method of claim 1 , wherein the first and/or the second nucleic acid molecule binds to a part of the polynucleotide to be detected.
9 . The method of claim 6 , wherein the contacting comprises applying the composition to the target polynucleotide to be detected immobilized onto a detection chip.
10 . The method of claim 1 , wherein the composition for detecting the polynucleotide further comprises a third protein-nucleic acid complex comprising an Argonaute protein associated with a third nucleic acid molecule,
wherein the third nucleic acid molecule comprises a nucleic acid sequence complementary to a third target nucleic acid region in the polynucleotide to be detected,
wherein the third nucleic acid molecule is conjugated with a third fluorophore different from the first and the second fluorophore and generating a third fluorescent signal,
wherein (b) further comprises detecting the third fluorescent signal resulting from the contacting of (a), wherein the third fluorescent signal is a fluorescent signal generated by the third fluorophore conjugated with the third nucleic acid sequence bound to the third target nucleic acid region,
wherein (c) further comprises calculating time fraction of bound state for the third fluorescent signal according to formula:
Time fraction of bound state= T/P,
wherein T is a total time of sensing the third fluorescent signal during a detection time, and P is the detection time,
wherein (d) comprises determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than the first threshold value set for the first target nucleic acid region, the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than the second threshold value set for the first target nucleic acid region, and the calculated time fraction of bound state for the third fluorescent signal is equal to or higher than a third threshold value set for the third target nucleic acid region,
wherein each of the first, the second, and the third threshold values is a value in a range of from 0.03 to 0.12.
11 . The method of claim 7 , wherein the composition for detecting the polynucleotide further comprises a third protein-nucleic acid complex comprising an Argonaute protein associated with a third nucleic acid molecule,
wherein the third nucleic acid molecule comprises a nucleic acid sequence complementary to a third target nucleic acid region in the polynucleotide to be detected,
wherein the third nucleic acid molecule is conjugated with a third fluorophore different from the first and the second fluorophore and generating a third fluorescent signal,
wherein (b) further comprises detecting the third fluorescent signal resulting from the contacting of (a), wherein the third fluorescent signal is a fluorescent signal generated by the third fluorophore conjugated with the third nucleic acid sequence bound to the third target nucleic acid region,
wherein (c) further comprises calculating time fraction of bound state for the third fluorescent signal according to formula:
Time fraction of bound state= T/P,
wherein T is a total time of sensing the third fluorescent signal during a detection time, and P is the detection time,
wherein (d) comprises determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than the first threshold value set for the first target nucleic acid region, the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than the second threshold value set for the first target nucleic acid region, and the calculated time fraction of bound state for the third fluorescent signal is equal to or higher than a third threshold value set for the third target nucleic acid region,
wherein each of the first, the second, and the third threshold values is a value in a range of from 0.03 to 0.12.
12 . The method of claim 10 , wherein the composition for detecting the polynucleotide further comprises a fourth protein-nucleic acid complex comprising an Argonaute protein associated with a fourth nucleic acid molecule,
wherein the fourth nucleic acid molecule comprises a nucleic acid sequence complementary to a fourth target nucleic acid region in the polynucleotide to be detected,
wherein the fourth nucleic acid molecule is conjugated with a fourth fluorophore different from the first and the second fluorophore and generating a fourth fluorescent signal,
wherein (b) further comprises detecting the fourth fluorescent signal resulting from the contacting of (a), wherein the fourth fluorescent signal is a fluorescent signal generated by the fourth fluorophore conjugated with the fourth nucleic acid sequence bound to the fourth target nucleic acid region,
wherein (c) further comprises calculating time fraction of bound state for the fourth fluorescent signal according to formula:
Time fraction of bound state= T/P,
wherein T is a total time of sensing the forth fluorescent signal during a detection time, and P is the detection time,
wherein (d) comprises determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than the first threshold value set for the first target nucleic acid region, the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than the second threshold value set for the first target nucleic acid region, the calculated time fraction of bound state for the third fluorescent signal is equal to or higher than the third threshold value set for the third target nucleic acid region, and the calculated time fraction of bound state for the fourth fluorescent signal is equal to or higher than a fourth threshold value set for the fourth target nucleic acid region,
wherein each of the first, the second, the third, and the fourth threshold values is a value in a range of from 0.03 to 0.12.
13 . The method of claim 11 , wherein the composition for detecting the polynucleotide further comprises a fourth protein-nucleic acid complex comprising an Argonaute protein associated with a fourth nucleic acid molecule,
wherein the fourth nucleic acid molecule comprises a nucleic acid sequence complementary to a fourth target nucleic acid region in the polynucleotide to be detected,
wherein the fourth nucleic acid molecule is conjugated with a fourth fluorophore different from the first and the second fluorophore and generating a fourth fluorescent signal,
wherein (b) further comprises detecting the fourth fluorescent signal resulting from the contacting of (a), wherein the fourth fluorescent signal is a fluorescent signal generated by the fourth fluorophore conjugated with the fourth nucleic acid sequence bound to the fourth target nucleic acid region,
wherein (c) further comprises calculating time fraction of bound state for the fourth fluorescent signal according to formula:
Time fraction of bound state= T/P,
wherein T is a total time of sensing the fourth fluorescent signal during a detection time, and P is the detection time,
wherein (d) comprises determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than the first threshold value set for the first target nucleic acid region, the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than the second threshold value set for the first target nucleic acid region, the calculated time fraction of bound state for the third fluorescent signal is equal to or higher than the third threshold value set for the third target nucleic acid region, and the calculated time fraction of bound state for the fourth fluorescent signal is equal to or higher than a fourth threshold value set for the fourth target nucleic acid region,
wherein each of the first, the second, the third, and the fourth threshold values is a value in a range of from 0.03 to 0.12.