T7 RNA polymerase engineering and RNA amplification

Award Information
Agency:
Department of Health and Human Services
Branch
n/a
Amount:
$178,517.00
Award Year:
2004
Program:
STTR
Phase:
Phase I
Contract:
1R41GM072412-01
Award Id:
71677
Agency Tracking Number:
GM072412
Solicitation Year:
n/a
Solicitation Topic Code:
n/a
Solicitation Number:
n/a
Small Business Information
2130 WOODWARD STREET, AUSTIN, TX, 78744
Hubzone Owned:
N
Minority Owned:
N
Woman Owned:
N
Duns:
n/a
Principal Investigator:
LIANGJINGCHEN
(512) 651-0200
LCHEN@AMBION.COM
Business Contact:
RIGOVALLEJO
(512) 651-0200
RVALLEJO@AMBION.COM
Research Institute:
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON

UNIVERSITY OF TEXAS HLTH SCI CTR
Austin, TX, 77225

Nonprofit college or university
Abstract
DESCRIPTION (provided by applicant): Microarrays are powerful tools that can impact new diagnostic procedures and expedite the drug development process. A significant but largely unrealized application is gene expression analysis from RNAlimited samples, such as blood, needle biopsies, laser capture microdissection (LCM) samples, and even single cells. The most widely used T7 RNA polymerase-based amplification method currenly lacks the necessary sophistication to meet these emerging needs. To overcome these limitations, we propose to increase the efficiency of T7 RNA amplification using an innovative protein engineering approach. Through combined rational design and random mutagenesis, mutant library selection and screening, we aim to identify a hyperactive T7 RNA polymerase that is 4-5 times more kinetically proficient than the current enzyme. We will expand this novel approach in phase II to include the discovery of mutant T7 polymerase enzymes that are both more thermostable (and thus extend high efficiency transcription for longer times, creating more product) and can incorporate biotinylated and Cy-modified nucleotides 5- to 10-fold more efficiently. Taken together, these improvements will lower the demand for input RNA by approximately 20-fold, and reduce the expense of modified nucleotides by as much as an order of magnitude.

* information listed above is at the time of submission.

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