Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing

Patent metadata

Field	Value
Publication number	US-2017145496-A1
Application number	US-201615357966-A
Country	US
Kind code	A1
Filing date	Nov 21, 2016
Priority date	Nov 20, 2015
Publication date	May 25, 2017
Grant date	—

Abstract

Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

First claim

1 - 50 . (canceled)

51 . A labeled nucleotide analog comprising:
a first avidin protein having four subunits, each subunit comprising one biotin binding site; a first nucleotide compound, bound to the first avidin protein, the first nucleotide compound comprising a polyphosphate element, a nucleoside element, an optional multivalent central core element, a terminal coupling element, and a nucleotide linker element; and a first dye-labeled compound, bound to the first avidin protein, the first dye-labeled compound comprising a donor dye, an acceptor dye, a terminal coupling element, a dye compound linker element, and a shield element.

52 - 54 . (canceled)

55 . The labeled nucleotide analog of claim 51 , wherein the donor dye and the acceptor dye are fluorescent dyes.

56 . The labeled nucleotide analog of claim 51 , wherein the first dye-labeled compound comprises at least two donor dyes.

57 . The labeled nucleotide analog of claim 56 , wherein the first dye-labeled compound comprises at least four donor dyes.

58 . The labeled nucleotide analog of claim 51 , wherein the first dye-labeled compound comprises at least two acceptor dyes.

59 . The labeled nucleotide analog of claim 58 , wherein the first dye-labeled compound comprises at least four acceptor dyes.

60 . The labeled nucleotide analog of claim 51 , wherein the first dye-labeled compound comprises at least two donor dyes and at least two acceptor dyes.

61 . The labeled nucleotide analog of claim 51 , wherein the dye compound linker element comprises a shield element or a side chain element.

62 . The labeled nucleotide analog of claim 51 , wherein the shield element of the first dye-labeled compound decreases photodamage of the first dye-labeled compound or of a biomolecule associated with the analog or increases brightness of the analog.

63 - 133 . (canceled)

134 . The labeled nucleotide analog of claim 51 , wherein the first dye-labeled compound is a compound of structural formula (IIIG):

wherein
each L′ is independently a dye compound linker element;
each S is independently a shield element;
each Dye is independently either an acceptor dye or a donor dye;
each B″ is independently a terminal coupling element;
each p is independently 0 or 1; and
each r″ is independently an integer from 0 to 8;
s is an integer from 1 to 6; and
t is 0 or 1;

wherein the compound comprises at least one shield element, at least one acceptor dye, and at least one donor dye.

135 . The labeled nucleotide analog of claim 134 , wherein each r″ is independently an integer from 0 to 4.

136 . The labeled nucleotide analog of claim 135 , wherein each r″ is independently an integer from 0 to 2.

137 . The labeled nucleotide analog of claim 134 comprising at least two donor dyes.

138 . The labeled nucleotide analog of claim 137 comprising at least four donor dyes.

139 . The labeled nucleotide analog of claim 134 comprising at least two acceptor dyes.

140 . The labeled nucleotide analog of claim 139 , comprising at least four acceptor dyes.

141 . The labeled nucleotide analog of claim 134 comprising at least two donor dyes and at least two acceptor dyes.

142 . The labeled nucleotide analog of claim 134 , wherein at least one dye compound linker element comprises a shield element or a side chain element.

143 . The labeled nucleotide analog of claim 134 , wherein the at least one shield element comprises a plurality of side chains.

144 . The labeled nucleotide analog of claim 143 , wherein at least one side chain has a molecular weight of at least 300.

145 . The labeled nucleotide analog of claim 143 , wherein all of the side chains have a molecular weight of at least 300.

146 . The labeled nucleotide analog of claim 143 , wherein at least one side chain comprises a polyethylene glycol.

147 . The labeled nucleotide analog of claim 143 , wherein at least one side chain comprises a negatively-charged component.

148 . The labeled nucleotide analog of claim 147 , wherein the negatively-charged component comprises a sulfonic acid.

149 . The labeled nucleotide analog of claim 143 , wherein at least one side chain comprises a substituted phenyl group.

150 . The labeled nucleotide analog of claim 149 , wherein the at least one side chain comprises the structure:

wherein each x is independently an integer from 1 to 6.

151 . The labeled nucleotide analog of claim 150 , wherein each x is independently an integer from 1 to 4.

152 . The labeled nucleotide analog of claim 143 , wherein at least one side chain comprises a triazole.

153 . The labeled nucleotide analog of claim 143 , wherein at least one side chain comprises the structure:

154 . The labeled nucleotide analog of claim 134 , wherein the at least one shield element comprises the structure:

wherein each y is independently an integer from 1 to 6.

155 . The labeled nucleotide analog of claim 134 , wherein the at least one shield element comprises the structure:

156 . The labeled nucleotide analog of claim 134 , wherein the terminal coupling elements.

157 . The labeled nucleotide analog of claim 134 , wherein the terminal coupling element comprises a biotin moiety.

158 . The labeled nucleotide analog of claim 157 , wherein the terminal coupling element comprises a bis-biotin moiety.

159 . The labeled nucleotide analog of claim 134 , wherein the at least one acceptor dye or the at least one donor dye is a cyanine dye.

160 . The labeled nucleotide analog of claim 134 , wherein the first dye-labeled compound does not contain a nucleoside.

161 - 187 . (canceled)

Assignees

• Pacific Biosciences California Inc

Inventors

• Sebo Lubomir
• Shen Gene
• Yue Stephen
• Osuna Honey
• Lapin Yuri
• Brogley Louis
• Fedorov Andrei

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title

• C07H19/207

  the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide · CPC title

• C09B69/105

  containing a methine or polymethine dye · CPC title