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

A method for determining the level of a target nucleic acid from different nucleic acids in a fixed and permeabilized single cell, the method comprising:

• contacting a fixed and permeabilized single cell with a plurality of pairs of SNAIL oligonucleotide primers having specificity for different target nucleic acids under conditions permissive for a specific hybridization of the plurality of pairs of SNAIL oligonucleotide primers to the different target nucleic acids, wherein each pair of the plurality of pairs of SNAIL oligonucleotide primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of the SPO and the PO of each pair of the plurality of pairs of SNAIL oligonucleotide primers comprises a first complementarity region and the first complementarity region of the SPO and the first complementarity region of the PO of each pair of the plurality of pairs of SNAIL oligonucleotide are complementary to two adjacent sequences on one of the different target nucleic acids, wherein the first complementarity region of the SPO of each pair of the plurality of pairs of SNAIL oligonucleotide is CR1 and the first complementarity region of the PO of each pair of the plurality of pairs of SNAIL oligonucleotide is CR1′;
• and each of the SPO and the PO of each pair of the plurality of pairs of SNAIL oligonucleotide further comprises a second complementarity region, wherein the second complementarity region of the SPO of each pair of the plurality of pairs of SNAIL oligonucleotide is CR2 and the CR1 and the CR2 of the SPO of each pair of the plurality of pairs of SNAIL oligonucleotide are located adjacent to one another and the second complementarity region of the PO of each pair of the plurality of pairs of SNAIL oligonucleotide is CR2′ and the CR1′ and the CR2′ of the PO of each pair of the plurality of pairs of SNAIL oligonucleotide are located adjacent to one another;
• wherein the CR2′ is a split region of the PO of each pair of the plurality of pairs of SNAIL oligonucleotide such that the 5′ and the 3′ ends of the PO of one pair of the plurality of pairs of SNAIL oligonucleotide hybridize to the CR2 of the SPO of the one pair of the plurality of pairs of SNAIL oligonucleotide and are positioned directly adjacent to one another after said contacting a fixed and permeabilized single cell with a plurality of SNAIL oligonucleotide primers;
• washing the cell to remove the plurality of pairs of SNAIL oligonucleotide primers unbound to the different target nucleic acids;
• after the washing step, contacting the cell with a ligase such that the 5′ and the 3′ ends of the PO of the one pair of the plurality of pairs of SNAIL oligonucleotide hybridized to the CR2 of the SPO of the one pair of the plurality of pairs of SNAIL oligonucleotide are ligated to each other and generating a closed circle;
• performing rolling circle amplifications in situ using the closed circle as a template and the SPO of the one pair of the plurality of pairs of SNAIL oligonucleotide 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 from the different nucleic acids in the fixed and permeabilized single cell by determining 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.