Title:
DNA-LINKED ENZYME-COUPLED ASSAYS
Document Type and Number:
WIPO Patent Application WO/2016/054024
Kind Code:
A3
Abstract:
Traditional enzyme characterization methods are low-throughput, and therefore limit engineering efforts in synthetic biology and biotechnology. Here we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. DLEnCA is generalizable for many enzymatic reactions, and here we adapt it for glucosyltransferases, methyltransferases, and oxidoreductases. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-Glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected post-reaction using qPCR or similar means of DNA analysis. For monitoring methyltransferases, consumption of SAM is observed by coupling to EcoRI methyltransferase. For monitoring oxidoreductases, consumption of NADH is observed by coupling to Taq or E. coli DNA ligase. OleD and two glucosyltransferases from Arabidopsis were used to verify the assay's generality toward glucosyltransferases. Two methyltransferases from human and Arabidopsis were used to verify the assay's generality towards methyltransferases. We show DLEnCA' s utility by mapping out the substrate specificity for these enzymes and observing the multiple steps of a biosynthetic pathway.
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Inventors:
ANDERSON JOHN CHRISTOPHER (US)
DE RAAD MARKUS (US)
DE RAAD MARKUS (US)
Application Number:
PCT/US2015/052920
Publication Date:
June 30, 2016
Filing Date:
September 29, 2015
Export Citation:
Assignee:
UNIV CALIFORNIA (US)
SUKOVICH DAVID J (US)
MODAVI CYRUS (US)
SUKOVICH DAVID J (US)
MODAVI CYRUS (US)
International Classes:
C12N9/00
Domestic Patent References:
| WO2000050630A2 | 2000-08-31 |
Foreign References:
| US20050118665A1 | 2005-06-02 | |||
| US20020155489A1 | 2002-10-24 |
Attorney, Agent or Firm:
MANN, Jeffry, S. et al. (Lewis & Bockius LLPOne Market, Spear Street Towe, San Francisco CA, US)
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