Multiplexed single molecule RNA visualization with a two-probe proximity ligation system

Patent metadata

Field	Value
Publication number	US-11008608-B2
Application number	US-201716079017-A
Country	US
Kind code	B2
Filing date	Feb 24, 2017
Priority date	Feb 26, 2016
Publication date	May 18, 2021
Grant date	May 18, 2021

Abstract

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.

First claim

What is claimed is:

1. A method for determining the level of a target nucleic acid in a single cell, the method comprising:
i) contacting a fixed and permeabilized cell with a pair of SNAIL oligonucleotide primers under conditions permissive for specific hybridization of the pair of SNAIL oligonucleotide primers to the target nucleic acid, wherein the pair of SNAIL oligonucleotide primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprises a first complementarity region complementary to adjacent sequences on the target nucleic acid, wherein the first complementarity region of SPO is CR1 and the first complementarity region of PO is CR1′; and each of SPO and PO further comprises a second complementarity region, wherein the second complementarity region of SPO is CR2 located adjacent to CR1 and the second complementarity region of PO is CR2′ located adjacent to CR1′, wherein CR2′ is a split region of PO such that after said contacting a fixed and permeabilized cell with the pair of SNAIL oligonucleotide primers, the 5′ and the 3′ ends of PO hybridize to CR2 and the 5′ and the 3′ ends of PO are positioned on CR2 directly adjacent to one another, wherein the fixed and permeabilized cell is a single cell;
ii) washing the cell to remove the pair of SNAIL oligonucleotide primers unbound to the target nucleic acid,
iii) after the washing step, contacting the cell with a ligase such that the 5′ and the 3′ ends of the PO hybridized to the target nucleic acid are ligated to each other and generating a closed circle;
iv) performing a rolling circle amplification using the closed circle as a template and SPO as a primer and generating an amplification product;
v) contacting the amplification product with a detection probe under conditions permissive for specific hybridization of the detection probe to the amplification product; and
vi) determining the level of the target nucleic acid in the single cell by detecting the level of the detection probe bound to the amplification product.

2. The method of claim 1 , wherein the pair of SNAIL oligonucleotide primers are denatured by heating the pair of SNAIL oligonucleotide primers before step i).

3. The method of claim 1 , wherein the cell is present in a population of cells.

4. The method of claim 3 , wherein the population of cells comprises a plurality of types of cells.

5. The method of claim 1 , further comprises contacting said fixed and permeabilized cell with a plurality of different pairs of SNAIL oligonucleotide primers having binding specificity for different target nucleic acids in the cell wherein each primer pair of different pairs of SNAIL oligonucleotide primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO of the each primer pair comprises a first complementarity region complementary to adjacent sequences on a target nucleic acid of the different target nucleic acids in the cell, wherein the first complementarity region of SPO is CR1 and the first complementarity region of PO is CR1′; and each of SPO and PO of the each primer pair further comprises a second complementarity region, wherein the second complementarity region of SPO is CR2 located adjacent to CR1 and the second complementarity region of PO is CR2′ located adjacent to CR1′, wherein CR2′ is a split region of PO such that after said contacting a fixed and permeabilized cell with the plurality of different pairs of SNAIL oligonucleotide primers, the 5′ and the 3′ ends of PO of the each primer pair hybridize to CR2 of SPO of the same primer pair of the plurality of different pairs of SNAIL oligonucleotide primers and the 5′ and the 3′ ends of PO of the each primer pair are positioned on CR2 of the same primer pair of the plurality of different pairs of SNAIL oligonucleotide primers directly adjacent to one another; and wherein the first complementarity region of each of SPO and PO of each primer pair of the plurality of different pairs of SNAIL oligonucleotide primers is complementary to a different target nucleic acid in the cell.

6. The method of claim 5 , wherein said plurality of different pairs of SNAIL oligonucleotide primers comprises at least 5 different pairs.

7. The method of claim 1 , wherein the target nucleic acid is RNA.

8. The method of claim 7 , wherein the RNA is mRNA.

9. The method of claim 1 , wherein the target nucleic acid is DNA.

10. The method of claim 1 , further comprising a step of contacting said fixed and permeabilized cell with marker detection reagents specifically binding to one or more markers in the cell.

11. The method of claim 10 , wherein the one or more markers are protein markers.

12. The method of claim 1 , wherein the detection probe is labeled with one or more of a fluorophore, an isotope, or a mass tag.

13. The method of claim 1 , wherein the detection probe is labeled with a fluorescent tag and the detecting step is performed by a fluorescence and confocal microscopy.

14. The method of claim 1 , wherein the sequence of the Padlock Oligonucleotide (PO) further comprises a barcode sequence.

15. The method of method of claim 1 , further comprising removing the detection probe after step vi).

16. The method of claim 1 , further comprising contacting said fixed and permeabilized cell with a plurality of different pairs of SNAIL oligonucleotide primers wherein each primer pair of the plurality of different pairs of SNAIL oligonucleotide primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO of the each primer pair comprises a first complementarity region complementary to adjacent sequences on the target nucleic acid, wherein the first complementarity region of SPO is CR1 and the first complementarity region of PO is CR1′; and each of SPO and PO further comprises a second complementarity region, wherein the second complementarity region of SPO is CR2 located adjacent to CR1 and the second complementarity region of PO is CR2′ located adjacent to CR1′, wherein CR2′ is a split region of PO such that after said contacting a fixed and permeabilized cell with the plurality of different pairs of SNAIL oligonucleotide primers, the 5′ and the 3′ ends of PO of the each primer pair hybridize to CR2 of the same primer pair of the plurality of different pairs of SNAIL oligonucleotide primers and the 5′ and the 3′ ends of PO of the each primer pair are positioned on CR2 of the same primer pair of the plurality of different pairs of SNAIL oligonucleotide primers directly adjacent to one another; and wherein the first complementarity region of each SPO and PO of each primer pair of the plurality of different pairs of SNAIL oligonucleotide primers is complementary to a different region of the target nucleic acid.

Assignees

• Univ Leland Stanford Junior

Inventors

• Samusik Nikolay
• Bava Felice Alessio
• Goltsev Yury
• Nolan Garry P

Classifications

• C12Q2533/107

  Probe or oligonucleotide ligation · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/682

  Signal amplification · CPC title