Highly specific circular proximity ligation assay
Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.
1. A method for sample analysis comprising:
(a) incubating a sample of blood plasma or serum comprising a target analyte that is at a concentration of less than 100 nM with:
(i) a first conjugate comprising a binding agent and a first splint oligonucleotide, and
(ii) a second conjugate comprising a binding agent and a second splint oligonucleotide,
wherein the first and second conjugates are a polyclonal antibody that has been affinity purified, split into a first portion and a second portion, and then conjugated to the first and second splint oligonucleotides,
under conditions suitable for binding of the binding agents of the first and second conjugates to the target analyte, to produce a product;
(b) incubating at least some of the product of step (a) with:
(i) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides; and
(ii) a ligase;
to produce a reaction mix comprising covalently closed circular molecules;
(c) treating at least some of the reaction mix of step (b) with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule; and
(d) after step (c), quantifying the amount of covalently closed circular molecules produced in step (b).
2. The method of claim 1 , wherein the first and second conjugates bind to the target analyte with a low affinity.
3. The method of claim 1 , wherein the quantifying step (d) is done by quantitative PCR, digital PCR, by hybridization to a microarray or by sequencing.
4. The method of claim 3 , wherein the primers used for the quantitative PCR target the ligation junctions in the covalently closed circular molecules of (d).
5. The method of claim 1 , wherein step (d) comprises amplifying the covalently closed circular molecules by rolling circle amplification (RCA) to produce RCA products.
6. The method of claim 5 , wherein the method comprises counting the RCA products.
7. The method of claim 1 , wherein the reactions of steps (a)-(c) are done in the same vessel.
8. The method of claim 7 , wherein step (b) comprises adding the set of probes and ligase to the vessel comprising the product of step (a), and step (c) comprises adding one or more exonucleases to the vessel comprising the ligation product of step (b).
9. The method of claim 1 , wherein:
(i) the sample comprises a plurality of target analytes,
(ii) step (a) comprises incubating the sample with multiple pairs of said first and second conjugates, wherein each pair of conjugates binds to a different target analyte; and
(iii) step (d) comprises quantifying the number of covalently closed circular molecules corresponding to each target analyte.
10. The method of claim 1 , wherein the target analyte is a protein.
11. The method of claim 1 , wherein:
at least two members of the set of probes of step (b)(i) each have a molecular index; and
step (d) is done by i. sequencing the covalently closed circular molecules and then ii counting the number of covalently closed circular molecules using the index sequence.
12. The method of claim 1 , wherein the target analyte is a cytokine.