Nucleic acid-based linkers for detecting and measuring interactions
The invention provides compositions comprising nucleic acid complexes for use in monitoring binding interactions and in measuring association and/or dissociation kinetics, detecting analytes including low concentration analytes, and screening library members. In some instances, the nucleic acid complexes are double-stranded nicked nucleic acids comprising a scaffold nucleic acid hybridized to one or more oligonucleotides. In some instances, a first, a second, a third, and optionally a fourth oligonucleotide are linked to moieties that are known to interact with each other or which are suspected of interacting with each other or of interacting with a common moiety such as an analyte. Changes in topology of the complex are used to determine the binding interactions of the various binding partners.
1. A method comprising
providing a single-stranded scaffold nucleic acid which is capable of hybridizing to a plurality of single-stranded oligonucleotides comprising a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, and a third single-stranded oligonucleotide, wherein the first single-stranded oligonucleotide is linked to a first binding partner, the second single-stranded oligonucleotide is linked to a second binding partner, and the third single-stranded oligonucleotide is linked to a third binding partner,
forming nucleic acid complexes by incubating the single-stranded scaffold nucleic acid with the plurality of single-stranded oligonucleotides under conditions that allow for hybridization of the plurality of single-stranded oligonucleotides to the single-stranded scaffold nucleic acid and binding of the first binding partner, the second binding partner and the third binding partner to each other in the nucleic acid complexes, wherein binding of the first binding partner and the second binding partner to each other is a first molecular interaction that results in a first looped nucleic acid conformation in the nucleic acid complexes, binding of the second binding partner and the third binding partner to each other is a second molecular interaction that results in a second looped nucleic acid conformation in the nucleic acid complexes, binding of the first binding partner and the third binding partner is a third molecular interaction that results in a third looped nucleic acid conformation in the nucleic acid complexes, and binding of the first binding partner, the second binding partner and the third binding partner is a fourth molecular interaction that results in a fourth looped nucleic acid conformation in the nucleic acid complexes, wherein each of the first molecular interaction, the second molecular interaction, the third molecular interaction, and the fourth molecular interaction is an intramolecular interaction, and
detecting the presence of the first nucleic acid conformation, the second nucleic acid conformation, the third nucleic acid conformation and/or the fourth looped nucleic acid conformation in the nucleic acid complexes by analyzing the nucleic acid complexes using gel electrophoresis,
wherein the first nucleic acid conformation, the second nucleic acid conformation, the third nucleic acid conformation and the fourth looped nucleic acid confirmation have different migration distances on a gel after the gel electrophoresis and are distinguishable from each other based on their migration distances through the gel.
2. The method of claim 1 , wherein each of the single-stranded oligonucleotides is about 60 nucleotides in length.
3. The method of claim 1 , wherein least one of the first binding partner, the second binding partner, and the third binding partner is a ligand, an antigen, or a receptor.
4. The method of claim 1 , wherein least one of the first binding partner, the second binding partner, and the third binding partner is a protein.
5. The method of claim 1 , wherein least one of the first binding partner, the second binding partner, and the third binding partner is an antibody or an antibody fragment.
6. The method of claim 1 , wherein the method further comprises measuring association and/or dissociation kinetics between (i) the first binding partner and the second binding partner, between (ii) the second binding partner and the third binding partner, between (iii) the first binding partner and the third binding partner, or among (iv) the first binding partner, the second binding partner and the third binding partner.
7. The method of claim 1 , wherein the method further comprises measuring association and/or dissociation kinetics among the first binding partner, the second binding partner, and the third binding partner.
8. The method of claim 1 , wherein the first molecular interaction, the second molecular interaction, the third molecular interaction, and the fourth molecular interaction comprise direct binding between the first binding partner and the second binding partner, direct binding between the second binding partner and the third binding partner, direct binding between the first binding partner and the third binding partner, and direct binding among the first binding partner, the second binding partner and the third binding partner, respectively.
9. The method of claim 1 , wherein the first molecular interaction, the second molecular interaction, the third molecular interaction, and the fourth molecular interaction comprise indirect binding between the first binding partner and the second binding partner, indirect binding between the second binding partner and the third binding partner, indirect binding between the first binding partner and the third binding partner and indirect binding among the first binding partner, the second binding partner and the third binding partner, respectively.
10. The method of claim 1 , wherein least one of the first binding partner, the second binding partner, and the third binding partner is a nucleic acid.
11. The method of claim 1 , wherein the first binding partner, the second binding partner, or the third binding partner is a bispecific receptor.