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Long lifetime alpha-hemolysin nanopores

a nanopore and alpha-hemolysin technology, applied in the field of alpha-hemolysin variants of staphylococcus aureaus, can solve the problems of difficult task, short time-consuming and laborious nanopores using wild-type alpha-hemolysins, and often serve as rate-limiting features of alpha-hemolysin nanopores during sequencing reactions, etc., to achieve more sequencing data than the effect of improving the lifetime of the nanopor

Inactive Publication Date: 2018-01-04
ROCHE SEQUENCING SOLUTIONS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a mutant version of a protein called staphylococcal alpha hemolysin (αHL) that can be added to a nanopore used for DNA sequencing. This mutant version improves the lifespan of the nanopore and allows for more sequencing data to be collected compared to using the wild-type protein. In simple terms, the mutant version of αHL makes the nanopore more durable and efficient for DNA sequencing.

Problems solved by technology

Previous work on DNA detection in the a-HL pore has focused on analyzing the ionic current signature as DNA translocates through the pore (Kasianowicz et al., 1996, Akeson et al., 1999, Meller et al., 2001), a very difficult task given the translocation rate (˜1 nt / μs at 100 mV) and the inherent noise in the ionic current signal.
While the use of nanopores has revolutionized DNA sequencing, nanopores using wild-type alpha-hemolysins are only able to generate sequence data for a short amount of time.
Hence, the lifetime of the alpha-hemolysin nanopore during the sequencing reaction often serves as the rate-limiting feature of the sequencing reaction.
Further, use of wild-type alpha hemolysin often results in a significant number of deletion errors, i.e., bases that are not measured.

Method used

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Examples

Experimental program
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Effect test

example 1

Expression and Recovery

[0136]This example illustrates the expression and recovery of protein from bacterial host cells, e.g., E. coli.

[0137]DNA encoding the wild-type a-HL was purchased from a commercial source. The sequence was verified by sequencing.

[0138]Plasmid Construction.

[0139]The gene encoding either a wild-type or variant α-hemolysin was inserted into a pPR-IBA2 plasmid (IBA Life Sciences, Germany) under the control of T7 promoter.

[0140]Transformation.

[0141]E. coli BL21 DE3 (from Life Technologies) cells were transformed with the expression vector comprising the DNA encoding the wild-type or variant α-hemolysin using techniques well-known in the art. Briefly, the cells were thawed on ice (if frozen). Next, the desired DNA (in a suitable vector / plasmid) was added directly into the competent cells (should not exceed 5% of that of the competent cells) and mixed by flicking the tube. The tubes were placed on ice for 20 minutes. Next, the cells were placed in a 42° C. water bat...

example 2

Alpha-Hemolysin Variants

[0147]The following example details the introduction of a mutation at a desired residue.

[0148]Mutations.

[0149]Site-directed mutagenesis is carried out using a QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) to prepare the example H35G+V149K+H144A (SEQ ID NO: 4) and H35G+E111N+M113A+126−131G+H144A+K147N (SEQ ID NO: 19), with the sequences including a C-terminal linker / TEV / HisTag for purification. QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) is also carried out to prepare a variant (E111N+M113A+126−131G+K147N, SEQ ID NO: 20) for Polymerase attachment, with the variant including a C-terminal SpyTag, KG linker, and HisTag. The variants were expressed and purified as in Example 1.

example 3

Assembly of Nanopore Including Variants

[0150]This example describes the assembly of a 1:6 heptameric nanopore including one subunit having a SpyTag sequence for subsequent polymerase attachment (the “α-HL-variant-SpyTag” subunit) and six α-HL-variant subunits with no SpyTag (the “α-HL-variant” subunits).

[0151]The α-HL-variant-SpyTag (E111N+M113A+126−131G+K147N, SEQ ID NO: 20)) was prepared and expressed as described in Examples 1 and 2 with a C-terminal SpyTag, KG linker, and HisTag. The α-HL-variant-SpyTag protein was then then purified on a cobalt affinity column using a cobalt elution buffer (200 mM NaCl, 300 mM imidazole, 50 mM tris, pH 8). The protein was stored at 4° C. if used within 5 days, otherwise 8% trehalose was added and stored at −80° C.

[0152]For the α-HL-variant subunits, variants of H35G+E111N+M113A+126-131G+H144A+K147N (SEQ ID NO: 19) were prepared and expressed as described in Examples 1 and 2 with a linker / TEV / HisTag and purified on a cobalt affinity column using...

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Abstract

Described herein are variants of alpha-hemolysin having at least one amino acid substitution at H35G, E111N, M113A, and / or K147N in the mature, wild-type alpha-hemolysin amino acid sequence. In certain examples, the variant may have a substitution at E111S, M113S, T145S, K147S, or L135I in the mature 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 an increased nanopore lifetime. Further, provided are variants that, in addition to providing increased lifetime, provide a decreased time-to-thread. Hence, the variants provided herein both increase nanopore lifetime and improve efficiency and accuracy of DNA sequencing reactions using nanopores comprising the variants.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 62 / 357,230, filed on Jun. 30, 2016, the contents of which are hereby incorporated in their entirety.SEQUENCE LISTING INCORPORATION BY REFERENCE[0002]This application hereby incorporates-by-reference a sequence listing submitted herewith in a computer-readable format, having a file name of 33725_WO_seqlist_ST25, created on Jun. 29, 2017, which is 56,233 bytes in size.TECHNICAL FIELD[0003]Disclosed are methods and compositions relating to Staphylococcus aureaus alpha-hemolysin variants that, when assembled into a multi-subunit nanopore, increase the sequencing lifetime of the nanopore during a nucleic acid sequencing reaction. Also disclosed are variants that, when assembled into a multi-subunit nanopore, improve sequencing efficiency and accuracy.BACKGROUND[0004]Hemolysins are members of a family of protein toxins that are produced by a wide variety of organisms. Some hemolysins, fo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68A61K38/02C12N15/01C07K14/00
CPCC12Q1/6869C07K14/00C12N15/01A61K38/02C07K14/31
Inventor AMBROSO, MARKCRAIG, TIMOTHYDIPIETRO, MATTHEWHARRIS, CORISSAPORTER, MARSHALL
Owner ROCHE SEQUENCING SOLUTIONS INC
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