IN VIVO EXPRESSION AND FUNCTION OF DNA ENZYMES

Yale University, New Haven CT

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Abstract

DESCRIPTION: 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.