Roche Sequencing Solutions, Inc.

Leland Stanford Junior University

Genia Technologies, Inc.

Yaozhong Zou

Mark R Ambroso

Corissa Harris

Marshall Winston Porter

Timothy Kellogg Craig

Matthew Dipietro

Brian K Kobilka

Timothy K Craig

Kapil Bajaj

Seong-Ho Shin

Giovanni Bellesia

Wiseley Wu

James Fairman

Alexxa Noble

Issa Delkaninia

Matt Dipetro

Timothy Craig

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

Alpha-hemolysin variants and uses thereof

Described herein are nanopore protein conjugates that can be used in DNA sequencing reactions. The nanopore protein conjugates includes a nanopore protein monomer that is joined to a DNA binding domain. The nanopore protein monomer is available to oligomerize with other nanopore protein monomers, while the DNA binding domain is available to bind to a template DNA strand. In certain examples, the nanopore protein monomer is an alpha-hemolysin monomer or variant thereof and the DNA binding domain is an Sso7d protein or variant thereof, such as an Sso7d-like protein. Also provided are nanopore protein assemblies incorporating the nanopore protein conjugates, along with methods of using the nanopore protein assemblies in sequencing reactions.

Nanopore protein conjugates and uses thereof

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. The &#x3b1;-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

Certain embodiments provide a GPCR fusion protein. In particular embodiments, the GPCR fusion protein comprises: a) a G-protein coupled receptor (GPCR); and b) an autonomously folding stable domain, where the autonomously folding stable domain is N-terminal to the GPCR and is heterologous to the GPCR. The GPCR fusion protein is characterized in that is crystallizable under lipidic cubic phase crystallization conditions. In certain embodiments, the GPCR fusion protein may be crystallizable in a complex with a G-protein or in a complex with an antibody that binds to the IC3 loop of the GPCR.

GPCR fusion protein containing an N-terminal autonomously folding stable domain, and crystals of the same

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Sunnyvale, CA, United States of America

Yaozhong Zou