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Structure guided rational engineering of novel DNA reagents.

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 4R42GM105097-02
Agency Tracking Number: R42GM105097
Amount: $761,592.00
Phase: Phase II
Program: STTR
Solicitation Topic Code: NIGMS
Solicitation Number: PA12-089
Timeline
Solicitation Year: 2013
Award Year: 2013
Award Start Date (Proposal Award Date): N/A
Award End Date (Contract End Date): N/A
Small Business Information
240 County Road
IPSWICH, MA -
United States
DUNS: 66605403
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 RICHARD MORGAN
 (978) 380-7485
 morgan@neb.com
Business Contact
 BRIAN TINGER
Phone: (978) 380-7485
Email: tinger@neb.com
Research Institution
 MOUNT SINAI SCHOOL OF MEDICINE
 
MOUNT SINAI HOSPITAL 1 GUSTAVE L LEVY PL
NEW YORK, NY 10029-6574
United States

 () -
 Domestic Nonprofit Research Organization
Abstract

DESCRIPTION (provided by applicant): Type II restriction enzymes (REases) are indispensible tools of modern medical research. It has long been a goal of REase manufacturers to be able to offer programmable specificity enzymes, where the sequence recognizedand resulting cutting activity can be precisely directed to any desired point in a DNA, as this will offer opportunity for improvement in many applications, from DNA sequencing to gene therapy. We have identified a new family of Type II endonucleases thatare amenable for the first time to the rational engineering of new DNA binding and cleavage specificities. To commercialize potentially thousands of new enzymes, we propose a structure-based approach, using crystallographic information to identify specificity determinants, which can then be rationally mutated to generate new nucleases with programmable specificities. In phase I of this application, we will prepare large amounts of several MmeI family enzymes, with the goal of having in hand well-diffracting crystals for two MmeI-family enzymes. In preliminary studies we have obtained well-diffracting cocrystals of MmeI that are highly suitable for structure determination. In Phase II, we will first determine structures for two MmeI family enzymes bound to DNA and then, as part of aim 2, use that information together with structure-based amino acid sequence alignments to generate a code of position-specific amino acids for the engineering of nucleases with programmable specificities. In aim 3, we will generate the potentially thousands of new specificity enzymes using site-directed mutagenesis protocols and, in aim 4, refine our understanding of specificity within this novel family of enzymes through structures of select mutants. An important application of MmeI-like enzymes is in technologies such as Serial Analysis of Gene Expression (SAGE) and paired-end sequence reads in next-generation DNA sequencing methods. Thus, as part of aim 5, we will use the structural information to engineer enzymes with extended reach between the recognition and cleavage sites for improving the quality of SAGE data and for speeding the assembly of genomes in DNA sequencing methods. Engineered Mme-I-like enzymes also offer the potential for targeted therapeutic use with minimal offtarget cleavage and toxicity. In aim 6, we will use our structural information to generate rare cutting MmeI-like endonucleases for therapeutic use. PUBLIC HEALTH RELEVANCE Type II restriction enzymes (REases) are indispensible tools of modern medical research. We propose to generate and commercialize thousands of new REases with programmable specificities for applications ranging from DNA sequencing to gene therapy.

* Information listed above is at the time of submission. *

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