As currently understood, biological catalysis is performed by protein enzymes or in some cases by enzymes that are made of RNA. In contrast, DNA has not been found to function as a catalyst in living organisms, where it exists primarily in double-stranded form. Recent breakthroughs in the in vitro evolution /DNA can also form higher- ordered structures and catalyze chemical transformations. These findings raise profound questions concerning the division of labor between the major biopolymers in modern living systems. Do natural organisms tap into the latent catalytic potential of DNA, or is DNA reserved exclusively for use as a medium for the storage of genetic information? A number of artificial enzymes made of DNA have been created using both rational design and combinatorial methods, some that operate under simulated physiological conditions. These advances in enzyme engineering have made possible, for the first time, the examination of the catalytic function of DNA enzymes in vivo. Specifically, an RNA- cleaving DNA metalloenzyme will be expressed in vivo using a novel single-stranded DNA expression vector. This vector will be derived from a natural bacterial retron that can be engineered to express foreign DNA molecules both in prokaryotic and eukaryotic cells. The DNA enzyme which can be engineered to specifically cleave any cellular RNA, will be designed to target several different mRNAs for destruction, thereby inhibiting the expression of specified genes. This strategy offers an alternative to current efforts that are striving to use RNA-cleaving ribozymes as anti-cancer and anti-viral agents. The increased chemical stability of DNA, for example, offers a significant advantage over RNA and protein enzymes for a variety of potential therapeutic and diagnostic applications. In addition, this work will introduce living organisms to a class of biocatalysts those that are made of DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM057500-01
Application #
2601459
Study Section
Biochemistry Study Section (BIO)
Project Start
1998-08-01
Project End
2001-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Yale University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Alexander, Matthew D; Burkart, Michael D; Leonard, Michael S et al. (2006) A central strategy for converting natural products into fluorescent probes. Chembiochem 7:409-16
Colby, David A; Chamberlin, A Richard (2006) Pharmacophore identification: the case of the ser/thr protein phosphatase inhibitors. Mini Rev Med Chem 6:657-65
Sreedhara, Alavattam; Li, Yingfu; Breaker, Ronald R (2004) Ligating DNA with DNA. J Am Chem Soc 126:3454-60
Emilsson, Gail Mitchell; Nakamura, Shingo; Roth, Adam et al. (2003) Ribozyme speed limits. RNA 9:907-18
Breaker, Ronald R; Emilsson, Gail Mitchell; Lazarev, Denis et al. (2003) A common speed limit for RNA-cleaving ribozymes and deoxyribozymes. RNA 9:949-57
Emilsson, G M; Breaker, R R (2002) Deoxyribozymes: new activities and new applications. Cell Mol Life Sci 59:596-607
Breaker, Ronald R (2002) Engineered allosteric ribozymes as biosensor components. Curr Opin Biotechnol 13:31-9
Li, Y; Breaker, R R (2001) In vitro selection of kinase and ligase deoxyribozymes. Methods 23:179-90
Li, Y; Liu, Y; Breaker, R R (2000) Capping DNA with DNA. Biochemistry 39:3106-14
Li, Y; Breaker, R R (1999) Phosphorylating DNA with DNA. Proc Natl Acad Sci U S A 96:2746-51

Showing the most recent 10 out of 11 publications