University of California

Harvard College

Agilent Technologies, Inc.

Other

Government of the United States of America, as Represented by the Secretary of Commerce

Mark Albert Akeson

David W Deamer

Daniel Branton

William Dunbar

Noah A Wilson

Roger Jinteh Arrigo Chen

Hugh Edward Olsen

Jeffrey Robert Sampson

Miten Jain

Seico Benner

Kathy Lieberman

Wenonah Vercoutere

Kate Lieberman

Logan Mulroney

George M Church

Bakthan Singaram

Rebecca L Braslau

Manuel Ares, Jr

Jenny Vo

Derek Steiner

Frank Cappuccio

David H Haussler

John J Kasianowicz

Felix Olasagasti

Jeffrey Matthew Nivala

Richard Baldarelli

Robin Abu-Shumays

Nicholas Hurt

Douglas Benjamin Marks

Felix A Olasagasti

Stephen Winters-Hilt

Gerald Maxwell Cherf

Seiko L Endo

Christopher Evan Lam

Kathy R Lieberman

William B Dunbar

Douglas B Marks

Jeffrey M Nivala

Roger Jinteh Chen

Michael Doody

Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

Compositions, devices, systems, and methods for using a nanopore

Provided are methods of adding a polymer of non-canonical nucleotides to the 3' end of a ribonucleic acid (RNA). In certain embodiments, the methods comprise combining an RNA, a polynucleotide-3′ nucleotidyl transferase, and non-canonical nucleotides, in a reaction mixture under conditions in which the polynucleotide-3′ nucleotidyl transferase adds a polymer of the non-canonical nucleotides to the 3′ end of the RNA. Such methods may further include analyzing the RNA using a nanopore. According to some embodiments, the methods include identifying the polymer of non-canonical nucleotides added to the 3′ end of the RNA, and determining the junction between the 3′ end of the RNA and the polymer of non-canonical nucleotides to identify the 3′ end of the RNA. Kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

Methods of adding polymers to ribonucleic acids

Provided are methods of analyzing capped ribonucleic acids (RNAs). The methods include translocating an adapted RNA through a nanopore of a nanopore device. The adapted RNA includes an RNA region, a 5' cap, and an adapter polynucleotide attached to the 5′ cap. The methods include monitoring ionic current through the nanopore during the translocating, translocating the 5′ cap through the nanopore, and identifying one or more ionic current features characteristic of the 5′ cap (e.g., a triphosphate linkage between the 5′ cap and nucleotide N1 of the RNA region, a 5′ to 5′ orientation of the 5′ cap and nucleotide N1 of the RNA region, and/or the like), translocating through the nanopore. Also provided are computer-readable media, computer devices, and systems that find use, e.g., in practicing the methods of the present disclosure.

Methods of analyzing capped ribonucleic acids

Provided are methods of producing size-selected nucleic acid libraries. The methods include contacting a nucleic acid sample and a nucleic acid binding reagent including an affinity tag, under conditions in which nucleic acids of less than a desired length are substantially bound to the nucleic acid binding reagent and nucleic acids of the desired length are substantially not bound to the nucleic acid binding reagent. The conditions include the duration of the contacting, the concentration of the nucleic acid binding reagent, or both. The methods further include separating, using the affinity tag, the nucleic acids of less than the desired length bound to the nucleic acid binding reagent from the nucleic acids of the desired length not bound to the nucleic acid binding reagent, to produce a size-selected nucleic acid library. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure, are also provided.

Methods of producing size-selected nucleic acid libraries and compositions and kits for practicing same

Provided are methods of adding a polymer of non-canonical nucleotides to the 3′ end of a ribonucleic acid (RNA). In certain embodiments, the methods comprise combining an RNA, a polynucleotide-3′ nucleotidyl transferase, and non-canonical nucleotides, in a reaction mixture under conditions in which the polynucleotide-3′ nucleotidyl transferase adds a polymer of the non-canonical nucleotides to the 3′ end of the RNA. Such methods may further include analyzing the RNA using a nanopore. According to some embodiments, the methods include identifying the polymer of non-canonical nucleotides added to the 3′ end of the RNA, and determining the junction between the 3′ end of the RNA and the polymer of non-canonical nucleotides to identify the 3′ end of the RNA. Kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

Described herein is a device and method for translocating a protein through a nanopore and monitoring electronic changes caused by different amino acids in the protein. The device comprises a nanopore in a membrane, an amplifier for providing a voltage between theside and trans side of the membrane, and an NTP driven unfoldase which processed the protein to be translocated. The exemplified unfoldase is the ClpX unfoldase from

Nanopore sensor for enzyme-mediated protein translocation

Disclosed are methods for polynucleotide sequencing that detect the location of selected nucleobases with greater precision. The methods can be used to determine the location and nature of modified bases in a polynucleotide, that is, non-canonical bases, or to improve accuracy of sequencing of 'problem' regions of DNA sequencing such as homopolymers, GC rich areas, etc. The sequencing method exemplified is nanopore sequencing. Nanopore sequencing is used to generate a unique signal at a point in a polynucleotide sequence where an abasic site (AP site, or apurinic or apyrimidinic site) exists. As part of the method, an abasic site is specifically created enzymatically using a DNA glycosylase that recognizes a pre-determined nucleobase species and cleaves the N-glycosidic bond to release only that base, leaving an AP site in its place.

Methods for determining base locations in a polynucleotide

Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (&#x2dc;&#x3e;50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

Nucleotide sequencing using an array of independently addressable nanopores

Methods for using a nanopore

Growing community of inventors

Santa Cruz, CA, United States of America

Mark Albert Akeson