Scaffold-based polymerase enzyme substrates
The invention provides a novel class of scaffold-based labeled polymerase enzyme substrates. The polymerase enzyme substrates have a multivalent core or scaffold to which is attached fluorescent dye moieties and nucleoside phosphate moities. The polymerase enzyme substrates have multiple fluorescent dye moities and/or multiple nucleoside phosphate moieties. Preferred multivalent cores comprise trifunctional six membered aromatic moities. The invention also provides for sequencing methods and kits with scaffold-based labeled polymerase enzyme substrates.
1. A composition comprising a covalent compound of the structure:
(DL D ) n -X-(L N PN) m
wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, each P is a polyphosphate moiety having the structure:
wherein u is from 2 to 10, each N is a nucleoside moiety, n is 2 or greater, m+n=4 or greater, and each L D and L N are either direct bonds or linkers.
2. The composition of claim 1 comprising a compound of the structure:
wherein D 1 and D 2 are each fluorescent dye moieties, L D1 and L D2 are each either direct bonds or linkers, each N is a nucleoside moiety, and m is two or greater.
3. The composition of claim 2 wherein D 1 is a FRET donor and D 2 is a FRET acceptor.
4. The composition of claim 1 wherein X comprises the structure:
5. The composition of claim l wherein X comprises the structure:
6. The composition of claim l wherein X comprises the structure:
wherein R comprises a linear or branched alkyl chain with from 1 to 10 carbons.
7. A sequencing mixture comprising a set of 4 nucleotide analog substrates, each having one of the bases A, G, C, T, or A, G, C, U, at least two of the substrates having the structure of claim 1 , and each substrate having at least one fluorescent dye moiety different from the fluorescent dye moieties on the other three substrates.
8. A method for nucleic acid sequencing comprising:
immobilizing a polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid;
exposing the immobilized polymerase enzyme complex to a plurality of labeled nucleotide analog substrates, at least one of the substrates comprising a compound having the structure of claim 1 ;
detecting the incorporation of substrates by observing fluorescence from the nucleotide analog substrates; and
using the detected incorporation over time to obtain sequence information about the template nucleic acid.
9. A composition comprising a covalent compound having the structure:
Y-(L-D-P—N) m
wherein Y is a multifunctional core having m linking sites, each L is either a direct bond or a linker, each D is a fluorescent dye moiety, each P is a polyphosphate having the structure:
wherein u is from from 2 to 10, each N is a nucleoside moiety, and m is greater than 2.
10. The composition of claim 9 wherein m is from 2 to about 10.
11. A composition comprising a compound having the structure:
wherein X is a multifunctional linker, A is a FRET acceptor dye moiety, each D is a FRET donor each L is a direct bond or a linker, each N is a nucleoside, each P is a polyphosphate having from 2 to 10 phosphates, m, n, s, t, p, and q are each independently either 1, 2, or 3.
12. The composition of claim 11 comprising a compound with the structure:
13. The composition of claim 12 wherein m, n, s, and t=1.
14. The composition of claim 11 wherein A comprises a cyanine dye.
15. The composition of claim 14 wherein each D comprises a cyanine dye.
16. A composition comprising a covalent compound of the structure:
(DL D ) n -X-(L N PN) m
wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, each P is a polyphosphate moiety having 2 to 10 phosphates, each N is a nucleoside moiety, n is 2 or greater, m+n=4 or greater, and each L D and L N are either direct bonds or linkers, wherein X comprises the structure:
17. A composition comprising a covalent compound of the structure:
(DL D ) n -X-(L N PN) m
wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, each P is a polyphosphate moiety having 2 to 10 phosphates, each N is a nucleoside moiety, n is 2 or greater, m+n=4 or greater, and each L D and L N are either direct bonds or linkers, wherein X comprises the structure:
18. A composition comprising a covalent compound of the structure:
(DL D ) n -X-(L N PN) m
wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, each P is a polyphosphate moiety having 2 to 10 phosphates, each N is a nucleoside moiety, n is 2 or greater, m+n=4 or greater, and each L D and L N are either direct bonds or linkers, wherein X comprises the structure:
19. A sequencing mixture comprising a set of 4 nucleotide analog substrates, each having one of the bases A, G, C, T, or A, G, C, U, at least two of the substrates having the structure:
(DL D ) n -X-(L N PN) m
wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, each P is a polyphosphate moiety having 2 to 10 phosphates, each N is a nucleoside moiety, n is 2 or greater, m+n=4 or greater, and each L D and L N are either direct bonds or linkers, wherein each substrate has at least one fluorescent dye moiety different from the fluorescent dye moieties on the other three substrates.
20. A method for nucleic acid sequencing comprising:
immobilizing a polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid;
exposing the immobilized polymerase enzyme complex to a plurality of labeled nucleotide analog substrates, at least one of the substrates comprising a compound having the structure:
(DL D ) n -X-(L N PN) m
wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, each P is a polyphosphate moiety having 2 to 10 phosphates, each N is a nucleoside moiety, n is 2 or greater, m+n=4 or greater, and each L 1 and L N are either direct bonds or linkers;
detecting the incorporation of substrates by observing fluorescence from the nucleotide analog substrates; and
using the detected incorporation over time to obtain sequence information about the template nucleic acid.