Cognate sampling kinetics
Provided are methods and compositions for measuring the transient binding of nucleotides and nucleotide analogs under conditions where the nucleotides or nucleotide analogs are unincorporable. The transient binding can be determined under single molecule observation conditions providing information about the kinetics of nucleotide analog sampling of the active site of the enzyme. The methods can be used for polymerase enzyme development, mechanistic understanding, and drug discovery.
1. A method comprising:
providing a polymerase enzyme complex comprising a template nucleic acid, a primer, and a polymerase enzyme under conditions wherein the interactions between a single polymerase and a labeled nucleotide analog can be monitored;
exposing the polymerase enzyme complex to one or more labeled nucleotide analogs in solution under conditions wherein the nucleotide analogs are unincorporatable;
observing the transient binding of the one or more labeled unincorporatable nucleotide analogs over time; and
using the observed transient binding to provide information about the enzyme, template nucleic acid, primer, or nucleotide analog or analogs.
2. The method of claim 1 wherein the primer is terminated, rendering the nucleotide analogs unincorporatable.
3. The method of claim 1 wherein the method is carried out with a plurality of nucleotide analogs, and the transient binding over time is used to determine the effect of nucleotide structure on enzyme performance.
4. A method for identifying a base in a template nucleic acid comprising:
providing a polymerase enzyme complex comprising a template nucleic acid, a primer, and a polymerase enzyme;
exposing the polymerase enzyme complex to one or more labeled nucleotide analogs in solution under conditions wherein the nucleotide analogs are unincorporatable, wherein one of the labeled unincornoratable nucleotide analogs is a cognate nucleotide analog; and
detecting multiple transient binding events corresponding to the binding or a labeled unincorporatable cognate nucleotide analog to the polymerase complex, thereby identifying the base corresponding to the cognate nucleotide analog in the template nucleic acid.
5. The method of claim 4 wherein the polymerase enzyme complex comprises a single polymerase molecule.
6. The method of claim 4 wherein the polymerase enzyme complex is immobilized on a substrate.
7. The method of claim 4 wherein the polymerase enzyme complex is immobilized in an optical confinement.
8. The method of claim 4 wherein at least a first and second primer are provided, wherein each of the primers are selected to bind to a known nucleotide sequence in which the identity of the base in the nucleotide sequence corresponding to the next cognate nucleotide is known and is different for the different primers; and wherein the polymerase enzyme complex is exposed to two or more labeled unincorporatable, nucleotide analogs, wherein the two or more labeled unincorporatable nucleotides comprise the next cognate nucleotides for the two primers.