Peilin Yu

Nucleotide modification at the γ-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase

The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their γ-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the γ-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.

Light-directed, programmable microarray synthesis

DNA Microarrays have become important in genome-wide expression studies. However, to fully use the power of the microarrays, more sophisticated and userfriendly data management and analysis tools have to be developed. We have developed a software package called MAT (Microarray Analysis Tools). MAT consists of a data entry tool, a web-based analysis tool and a relational database. User interfaces written in Java and JDBC are used to access the relational database (Sybase Adaptive server 11.5). The data entry tool is a form-based interface for user-friendly data entry. The web-based analysis tools consist of a synthetic image view tool, a graphic plot tool to display results from a series of experiments and a neural network-based data clustering tool. All of the interfaces are inter-connected. A user can view a single experiment or multiple experiments. A user can plot a time course, as well as a correlation graph with different experimental conditions. The mouse and human clone databases are automatically updated by searching through GenBank. The program also allows for the user to obtain GenBank data for any clone. Finally, the cluster tool will provide an advanced algorithm for clustering selected data. Our MAT tools are facilitating gene expression studies at AECOM, and can be adapted for use in other research laboratories.