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 labeled polymerase conjugate, comprising:
a polymerase linked to at least three labels to form a labeled polymerase conjugate,
wherein the conjugate has polymerase activity.
2 . The conjugate of claim 1 , wherein the conjugate comprises three, four, five, six, seven or more labels.
3 . The conjugate of claim 2 , wherein the polymerase activity is at least about 80% relative to the polymerase activity of the unconjugated polymerase.
4 . The conjugate of claim 2 , wherein the at least three labels are linked to an attachment site on the polymerase.
5 . The conjugate of claim 2 , wherein the at least three labels are linked to different attachment sites on the polymerase.
6 . The conjugate of claim 2 , wherein the polymerase further comprises a modification enzyme recognition sequence.
7 . The conjugate of claim 6 , wherein the modification enzyme recognition sequence is a biotin ligase modification site.
8 . A method for creating a labeled polymerase conjugate, comprising:
linking a polymerase to a plurality of labels to form a labeled polymerase conjugate having polymerase activity,
where the plurality of labels are linked to the polymerase.
9 . The method of claim 8 , wherein the linking further comprises linking the polymerase to at least three labels.
10 . The method of claim 8 , wherein the linking further comprises linking the plurality of labels to an attachment site on the polymerase.
11 . The method of claim 8 , wherein the linking further comprises linking the plurality of labels to independent attachment sites on the polymerase.
12 . The method of claim 8 , wherein the linking further comprises linking a polymerase including a modification enzyme recognition sequence to the plurality of labels.
13 . The method of claim 12 , wherein the modification enzyme recognition sequence is a biotin ligase modification site, and further comprising linking a biotin moiety to the polymerase to produce a biotinylated polymerase.
14 . The method of claim 13 , further comprising contacting the biotinylated polymerase with an avidin moiety linked to the plurality of labels under conditions where the avidin moiety binds to the biotin moiety.
15 . A labeled polymerase conjugate, comprising:
a polymerase linked to a plurality of labels to form a labeled polymerase conjugate,
where the conjugate has polymerase activity, and
where at least one label undergoes energy transfer with a labeled nucleotide bound to an active site of the polymerase.
16 . The conjugate of claim 15 , where the labeled nucleotide comprises a polyphosphate and where the nucleotide label is linked to the terminal phosphate of the polyphosphate.
17 . The conjugate of claim 15 , wherein the energy transfer produces a signal in a single molecule reaction.
18 . The conjugate of claim 15 , wherein the plurality of labels are connected to the polymerase at an attachment site on the polymerase.
19 . The conjugate of claim 15 , wherein the plurality of labels are connected to the polymerase at a plurality of attachment sites on the polymerase.
20 . The conjugate of claim 15 , wherein the plurality of labels includes three or more labels.
21 . The conjugate of claim 20 , wherein the polymerase comprises a modification enzyme recognition sequence.
22 . A method for generating a signal, comprising:
contacting a labeled polymerase conjugate including a polymerase linked to a label with a labeled nucleotide under conditions where the polymerase catalyzes the incorporation of the labeled nucleotide into a nucleic acid, and the label emits, or induces the emission of, a signal indicative of such nucleotide incorporation.
23 . The method of claim 22 , wherein the labeled polymerase conjugate further comprises at least three labels.
24 . The method of claim 22 , wherein the plurality of labels are attached to an attachment site on the polymerase.
25 . The method of claim 23 , wherein the plurality of labels are attached to independent attachment sites on the polymerase.
26 . The method of claim 23 , wherein the polymerase further comprises a modification enzyme recognition sequence.
27 . The method of claim 26 , wherein the modification enzyme recognition sequence further comprises a biotinylation site, and the polymerase comprises a biotin moiety.
28 . The method of claim 22 , further comprising detecting the signal.
29 . The method of claim 28 , further comprising analyzing the signal to identify the nucleotide that is incorporated.
30 . A labeled polymerase conjugate, comprising: a polymerase linked to one or more labels, where the conjugate has polymerase activity and emits upon continuous excitation a total photon count of at least 10 2 photons before irreversibly photobleaching.
31 . The conjugate of claim 30 , wherein the polymerase is linked to at least three labels.
32 . The conjugate of claim 31 , wherein the conjugate emits a total photon count of at least 10 8 photons as measured using a test detection system.
33 . A labeled polymerase conjugate, comprising:
a first member of a binding pair linked to a polymerase; and
a second member of the binding pair linked to at least one label,
where the first and the second member of the binding pair are linked to each other to form a labeled polymerase conjugate having polymerase activity.
34 . The conjugate of claim 33 , wherein the first member comprises a biotin moiety and the second member comprises an avidin moiety.
35 . The conjugate of claim 33 , wherein the second member is linked to at least three labels.
36 . The conjugate of claim 33 , wherein the at least one label generates a signal.
37 . The conjugate of claim 33 , wherein the at least one label is positioned to undergo FRET with a labeled nucleotide bound to the nucleotide binding site of the polymerase.