Identification of biomolecular interactions
The present disclosure, in some aspects, provides compositions, systems and methods for proximity-based detection of target biomolecules of interest.
1. A biomolecule detection system comprising
(a) a single-stranded nucleic acid comprising two 3′ ends, wherein one 3′ end comprises a first anchor domain and the other 3′ end comprises a second anchor domain; and
(b) a barcoded nucleic acid comprising a primer domain, a barcode domain and a nucleotide domain that is complementary to the first anchor domain of the single-stranded nucleic acid of (a).
2. The system of claim 1 , wherein the single-stranded nucleic acid is a conjugate comprising a first nucleic acid strand comprising a 5′ end linked to a 5′ end of a second nucleic acid strand.
3. The system of claim 2 , wherein the 5′ end of the first nucleic acid strand is covalently linked to the 5′ end of the second nucleic acid strand.
4. The system of claim 3 , wherein the single-stranded nucleic acid is produced by reacting an azide group at the 5′ end of the first nucleic acid strand with an alkyne group at the 5′ end of the second nucleic acid strand.
5. The system of claim 4 , wherein the single-stranded nucleic acid of (a) comprises two 5′ primer domains.
6. The system of claim 5 , further comprising a pair of primers, wherein one of the primers is complementary to one of the primer domains of the single-stranded nucleic acid, and the other primer is complementary to the other primer domain of the single-stranded nucleic acid.
7. The system of claim 1 , further comprising a nucleic acid adaptor that comprises (i) a unique molecular identifier and (ii) a nucleotide domain that is complementary to the second anchor domain of the single-stranded nucleic acid of (a).
8. The system of claim 1 , further comprising a binding biomolecule that binds to a target biomolecule of interest.
9. The system of claim 8 , wherein the binding biomolecule is a nucleic acid or a protein.
10. The system of claim 8 , wherein the binding biomolecule is an antibody.
11. The system of claim 1 , further comprising a polymerase, a ligase and/or a terminal transferase.
12. The system of claim 1 , further comprising a nucleic acid adaptor that comprises (i) a paired nucleotide sequence flanked by (ii) an unpaired primer domain and (iii) an unpaired homopolymer domain.
13. The system of claim 12 , wherein the nucleic acid adaptor is linked to biotin.
14. The system of claim 1 , further comprising at least two barcoded nucleic acids, wherein each barcoded nucleic acid comprises a primer domain, a barcode domain and a nucleotide domain that is complementary to the first anchor domain of the single-stranded nucleic acid of (a).
15. A method, comprising
combining in reaction solution that comprises polymerase and nucleoside triphosphates (NTPs) (a) a single-stranded nucleic acid comprising two 3′ ends, wherein each 3′ end comprises an anchor domain, (b) a barcoded nucleic acid comprising a primer domain, a barcode domain and a nucleotide domain that is complementary to one of the anchor domains of the single-stranded nucleic acid, wherein the at least one barcoded nucleic acid is linked to a binding biomolecule that binds to a target biomolecule of interest, and (c) a biomolecule comprising a nucleotide domain that is complementary to the other of the anchor domains of the single-stranded nucleic acid, thereby forming a reaction mixture; and
incubating the reaction mixture under conditions that result in binding of the single-stranded nucleic acid of (a) to the barcoded nucleic acid of (b) and to the biomolecule of (c), and extension of the single-stranded-nucleic acid through the primer domain of the barcoded nucleic acid, thereby producing a partially-double-stranded molecule.