Pacific Biosciences of California, Inc.

Lubomir Sebo

Stephen T Yue

Louis Brogley

Gene W Shen

Jeremiah Hanes

Yuri Lapin

Honey Osuna

Andrei Fedorov

Erik Miller

Satwik Kamtekar

Keith P Bjornson

John Lyle

Frank Zheng

Robin A Emig

Lei Jia

Ying Yang

Wei-Chuan Sun

Leewin Chern

Thomas Linsky

Jeffrey Wegener

Arek Bibillo

Natasha Popovich

Xiangxu Kong

Robert Joseph Weber

Stephen P Dudek

Duc Do

Zhong Wang

Grace Lee

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.

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

Protected dye-labeled reagents

Compositions, methods, and systems are provided for fluorescent polymerase enzyme substrates comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Fluorescent polymerase enzyme substrates of the invention have a protein shield between the fluorescent dye moieties and nucleotide moieties of the polymerase enzyme substrate. The polymerase enzyme substrates have a nucleotide component and a dye component, each attached to a protein. The attachments can be covalent. The protein can, for example, prevent the direct interaction of the fluorescent dye moiety with the enzyme when carrying out nucleotide synthesis, preventing photodamage to the enzyme. The polymerase enzyme substrates of the invention can have multiple dyes and multiple nucleotide moieties.

Fluorescent polymerase enzyme substrates having protein shields

Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds 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 compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.

Protected fluorescent reagent compounds

Modified nucleotide reagents

Compositions comprising covalently modified and mutated biotin-binding proteins, particularly biotin-binding proteins having a negative charge at physiological pH, are provided. Methods of producing such proteins are also provided, as are methods of immobilizing, sequencing, and making nucleic acids employing such proteins.

Modified biotin-binding proteins for immobilization

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Redwood City, CA, United States of America

Lubomir Sebo