US 12,338,490 B2
Multiplexed single molecule RNA visualization with a two-probe proximity ligation system
Nikolay Samusik, Mountain View, CA (US); Felice Alessio Bava, Menlo Park, CA (US); Yury Goltsev, Stanford, CA (US); and Garry P. Nolan, Redwood City, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Filed on Apr. 14, 2021, as Appl. No. 17/230,706.
Application 17/230,706 is a continuation of application No. 16/079,017, granted, now 11,008,608, previously published as PCT/US2017/019443, filed on Feb. 24, 2017.
Claims priority of provisional application 62/300,596, filed on Feb. 26, 2016.
Prior Publication US 2021/0238665 A1, Aug. 5, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12Q 1/682 (2018.01); C12Q 1/6841 (2018.01); G01N 33/542 (2006.01)
CPC C12Q 1/6841 (2013.01) [C12Q 1/682 (2013.01); G01N 33/542 (2013.01); C12Q 2525/307 (2013.01); C12Q 2531/125 (2013.01); C12Q 2533/107 (2013.01); C12Q 2543/10 (2013.01); C12Q 2600/16 (2013.01)] 14 Claims
OG exemplary drawing
 
1. A method for determining the level of a target nucleic acid in a fixed and permeabilized single cell, the method comprising:
contacting a fixed and permeabilized single cell with a pair of SNAIL oligonucleotide primers under conditions permissive for a specific hybridizations 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 comprise a first complementarity region and the first complementarity region of the SPO and the first complementarity region of the PO are complementary to two adjacent sequences on the target nucleic acid, wherein the first complementarity region of the SPO is CR1 and the first complementarity region of the PO is CR1′ and each of the SPO and the PO further comprises a second complementarity region, wherein the second complementarity region of the SPO is CR2 located adjacent to the CR1 and the second complementarity region of the PO is CR2′ located adjacent to the CR1′; wherein the CR2′ is a split region of the PO such that the 5′ and the 3′ ends of the PO hybridize to the CR2 and are positioned directly adjacent to one another after said contacting a fixed and permeabilized single cell with a pair of SNAIL oligonucleotide primers;
washing the cell to remove the pair of SNAIL oligonucleotide primers unbound to the target nucleic acid;
after the washing step, contacting the cell with a ligase such that the 5′ and the 3′ ends of the PO hybridized to the CR2 are ligated to each other and generating a closed circle;
performing in situ rolling circle amplification using the closed circle as a template and the SPO as a primer and generating an amplification product in situ;
contacting the amplification product in situ with a nucleic acid detection probe labeled with one or more of a fluorophore, an isotope, or a mass tag under conditions permissive for a specific hybridization of the detection probe to the amplification product and generating a labeled cell; and
determining the level of the target nucleic acid in the fixed and permeabilized single cell by detecting the level of the detection probe bound to the amplification product in the labeled cell and subjecting the labeled cell to an analysis performed by mass cytometry or fluorescence-activated flow cytometry.