enzymatic DNA and in vitro evolution
	Ronald Breaker, Ph.D. Henry Ford II Professor of Molecular, Cellular and Developmental Biology, HHMI Investigator Email: ronald.breaker@yale.edu Room: KBT 506 Phone: 432-9389/432-6554 Web site B.S. University of Wisconsin-Stevens Point 1987; Ph.D. Purdue University 1992

Ongoing investigations into the mechanisms of cellular life are revealing the details of many biological processes including information transfer, catalysis, signal transduction and molecular recognition. The fundamental roles of nucleic acids in the process of information transfer have been well established. However, the participation of RNA and DNA in other critical biochemical processes only now is becoming clearer. A significant advance in this area of research has been the discovery that RNA, like proteins, can function as an enzyme and catalyze chemical reactions. Ribozymes also may have played a critical role in the origin and evolutionary progression of life during the ?NA world? when an early metabolic state is believed to have been guided entirely by enzymes made of RNA. The sophistication of these primitive ribozymes and of modern ribozymes is defined by the structural and functional versatility of nucleic acids, and these parameters have yet to be fully explored.

We are probing the structural and catalytic repertoire of nucleic acids by using in vitro evolution? method by which rare RNAs or DNAs with new and improved functions can be isolated from pools of mutagenized or random-sequence molecules. In vitro evolution is modeled after the process of Darwinian evolution and is composed of iterative cycles of selection and amplification at the molecular level. Individual RNA or DNA molecules that display the desired phenotype are selected and subsequently amplified by chemical or enzymatic means. In related efforts, we are pioneering methods for the modular rational design of RNA and DNA enzymes that have new or improved catalytic function. For example, both modular rational design and in vitro evolution methods have been used to create a variety of allosteric ribozymes. These RNA-based molecular switches can be engineered to undergo activation or deactivation in the presence of small organic compounds, heavy metals, or even light.

The similarity in chemical structure between RNA and DNA had posed an important question: Can single-stranded DNA molecules, like proteins and RNA, assume distinct secondary and tertiary structures that function as catalysts? We have addressed this question by successfully screening a pool of 10 trillion different DNAs for molecules that catalyze their own self-destruction or the destruction of other target DNA molecules. In addition, we have created examples of DNA enzymes that use amino acids as cofactors, thereby demonstrating that nucleic acid enzymes can make use of the same chemical moieties used by proteins for the formation of enzyme active sites. Most recently, we have created a series of DNA enzymes that catalyze the reactions typically used for DNA cloning. We are continuing to define the catalytic potential of both RNA and DNA under physiological conditions and to explore the range of chemical reactions that can be catalyzed by novel nucleic acid enzymes when they are created outside the physical confines and conditions of the cell.

Selected Publications

N. Carmi, S. Balkhi and R. R. Breaker (1998) Cleaving DNA with DNA. Proc. Natl Acad. Sci. USA 95:2233-2237.

Y. Li and R. R. Breaker (1999) Phosphorylating DNA with DNA. Proc. Natl. Acad. Sci. USA 96: 2746-2751.

G. Soukup and R. R. Breaker (1999) Engineering Precision RNA Molecular Switches. Proc. Natl. Acad. Sci. USA 96:3584-3589.

M. Koizumi, G. A. Soukup, J. Q. Kerr and R. R. Breaker (1999) Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nature Struct. Biol. 6:1062-1071.

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