Labeled enzyme compositions, methods and systems
Disclosed herein are conjugates comprising a biomolecule linked to a label that have biological activity and are useful in a wide variety of biological applications. For example, provided herein are labeled polymerase conjugates including a polymerase linked to one or more labels, wherein the conjugate has polymerase activity. Such conjugates can exhibit enhanced biological activity and/or superior detectability as compared to conventional labeled polymerases. Also disclosed herein are improved methods for preparing such conjugates, and methods and systems for using such conjugates in biological applications such as nucleotide incorporation, primer extension and single molecule sequencing.
1. A method for creating a labeled polymerase conjugate, comprising:
linking a polymerase to at least three optically-detectable dye labels to form a labeled polymerase conjugate having polymerase activity and the at least three dye labels linked to the polymerase,
wherein the polymerase includes a biotin ligase modification site, and the linking comprises linking a biotin moiety to the polymerase to produce a biotinylated polymerase.
2. The method of claim 1 , wherein the linking comprises linking the polymerase to four, five, six, or seven optically-detectable dye labels.
3. The method of claim 1 , wherein the linking comprises linking the at least three optically-detectable dye labels to an attachment site on the polymerase.
4. The method of claim 1 , wherein the linking comprises linking the at least three optically-detectable dye labels to independent attachment sites on the polymerase.
5. The method of claim 1 , wherein the linking further comprises contacting the biotinylated polymerase with an avidin moiety linked to the at least three optically-detectable dye labels under conditions where the avidin moiety binds to the biotin moiety.
6. A method for creating a labeled polymerase conjugate, comprising:
linking a polymerase to at least three optically-detectable dye labels to form a labeled polymerase conjugate having polymerase activity and the at least three dye labels linked to the polymerase, wherein the polymerase includes one or more biotin acceptor peptide sequence.
7. The method of claim 6 , wherein at least one of the optically-detectable dye labels is linked to one biotin acceptor peptide sequence.
8. The method of claim 7 , wherein the at least three optically-detectable dye labels are linked to one biotin acceptor peptide sequence of the polymerase.
9. The method of claim 6 , wherein the at least three optically-detectable dye labels are linked to a plurality of biotin acceptor peptide sequences of the polymerase.
10. The method of claim 1 , wherein the labeled polymerase conjugate has activity that is at least about 80% relative to the polymerase activity of the unconjugated polymerase.
11. The method of claim 1 , wherein the labeled polymerase conjugate emits upon continuous excitation a total photon count of at least 10 2 photons before irreversibly photobleaching.
12. The method of claim 11 , wherein the labeled polymerase conjugate emits a total photon count of at least 10 6 photons as measured using a test detection system.
13. The method of claim 12 , wherein the labeled polymerase conjugate emits a total photon count of at least 10 8 photons as measured using a test detection system.
14. The method of claim 1 , wherein at least one of the linked optically-detectable dye labels is positioned to undergo FRET with a labeled nucleotide bound to the nucleotide binding site of the labeled polymerase conjugate.
15. The method of claim 14 , wherein at least three of the linked optically-detectable dye labels are positioned to undergo FRET with a labeled nucleotide bound to the nucleotide binding site of the labeled polymerase conjugate.