• Carlos F. Barbas, III, Ph.D.

    Kellogg Professor and Chair in Molecular Biology
    The Skaggs Institute for Chemical Biology
    and the Departments of Chemistry and Cell & Molecular Biology

  •   The Scripps Research Institute
    10550 North Torrey Pines Rd.
    La Jolla, CA 92037

  • Carlos F. Barbas, III, Ph.D. 
    Roberta  Fuller, Sr. Res. Assistant 
    Thom  Gaj, Ph.D. 
    Jarlath  Garcia 
    Xianxing  Jiang, Ph.D. 
    Brian  Lamb, Ph.D. 
    Robyn  Leary, Ph.D. 
    Jia  Liu, Ph.D. 
  • Mishelle  McClanahan-Shinn 
    Pedro  Perdigão 
    Bianca  Romana 
    Jingjing  Song 
    Mark  Wallen 
    Wei  Zhang, Ph.D. 
    Michael  Zorniak, Ph.D. 
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Protein-Like DNA Enzymes


Nucleic acid libraries provide tremendous opportunities for the selection of novel ligands and catalysts since the polymerase chain reaction, PCR, allows for the synthesis and selection of libraries containing more than 1014 different molecules. There are now many examples of nucleic acids that have been selected to bind proteins and small molecules and to catalyze a limited set of reactions. The catalytic and mechanistic scope of nucleic acids is limited since the natural nucleotide monomers possess minimal functionality compared to the repertoire available to Nature's dominant catalytic biopolymers, proteins. In recognition of this shortcoming, much attention has been focused on the development of functionalized nucleotides suitable for in vitro selection with the hope of increasing the potential of nucleic acids for binding and catalysis. Functionalized nucleotide triphosphates have been shown to be substrates for RNA polymerases and catalytic RNA's dependent on the modified base for their activity have been selected. Like RNA, DNA has also been selected to bind proteins and small molecules and more recently to catalyze reactions. While DNA possesses enhanced stability as compared to RNA, the lack of a 2'-hydroxyl group which provides for the enhanced stability of this molecule further reduces the functionality available to this molecule for chemistry. In contrast to the success achieved in identifying modified nucleotide triphosphates for RNA libraries, prior to our work there was but a single a deoxynucleotide triphosphate, 5-(1-pentynyl)-2'deoxyuridine triphosphate, that had been demonstrated to be a good substrate for a thermostable DNA polymerase and utilized in an in vitro DNA selection study. Indeed, difficulties in identifying modified deoxynucleotide triphophates substrates for the thermostable polymerases required for PCR have led recently to the development of novel strategies for in vitro selection without enzymatic amplification.

What if DNA could be endowed with the functionality of proteins? Might it be a more efficient and versatile catalyst?

 
Fig. 1. Making DNA a protein. Through synthesis and design we have dramatically expanded the chemical potential of DNA for binding and catalysis. Now protein-like DNAs can be prepared enzymatically as efficiently with modified dNTP's as natural DNA. Analogs of dTTP are shown. 

Through chemical synthesis we have developed a new class of DNA bases that can be used to construct protein-like DNA's (Figure 1). We synthesized novel functionalized deoxyuridine triphosphate derivatives that are good substrates for the DNA polymerases used to construct large libraries of nucleic acids, the reverse transcriptases and thermal stable polymerases. We have designed these bases to contain the side chains common to amino acids as well a novel side chains that proteins themselves don't possess.

Fig. 2. Protein-like DNA Specifically cleaves RNA.

In a collaborative effort with the Joyce group we have created the first protein-like DNA enzyme (Figure 2), a general purpose RNA cleaving enzyme that is superior to 'natural' DNA enzymes. This catalyst may be directly applicable as an antiviral agent. In the future we hope to create 'protein-like' DNA enzymes that perform the small-molecule chemistry of interest to synthetic chemists.


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