CROSS-LINKED ANTIGENE OLIGOMERS EFFECT ON TRANSCRIPTION

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In this MORE Faculty Development Award application, Dr. Norman S. Kondo, Professor of Chemistry, University of the District of Columbia (UDC) proposes to conduct research in the laboratory of Dr. Paul S. Miller, Professor of Biochemistry, Johns Hopkins University (JHU) for five consecutive summers. Through this effort Dr. Kondo plans to utilize newly acquired knowledge and expertise in nucleic acid biochemistry gained at JHU to establish a nucleic acid synthesis laboratory at UDC. In addition, a collaborative research relationship will also be established and continued with Dr. Miller beyond the grant period. The overall goal of the proposed research project is to determine the effect antigen oligonucleotides cross-linked to either or both strand/s of duplex DNA would have on its transcription. Antigen deoxynucleotides (pyrimidine-rich) complementary to the purine-rich (R) strand of the duplex DNA will be synthesized such that modified bases, designed to bind to pyrimidine interruptions in the R strand, are included. The 5'-end of the antigene oligonucleotide will be derivatized with a photoreactive psoralen residue and an amino-linker arm will be attached to the 3'-end (to reduce susceptibility to 3'-exonucleases. The selected target DNA will originate from purine-rich regions on the promoter site of the 92- kDa form of the collagenase IV gene. A DNA insert encompassing these regions will be synthesized by standard phosphoramidite chemistry and then inserted between T-7 and SP6 promoters in the vector, pGEM-3Z. After linearization of the plasmid, antigene oligomers will be directed to selected sites on the DNA target utilizing Hoogsteen type hydrogen bonding interactions. Ultraviolet irradiation of the triplex will cross- link the antigene oligonucleotide with the target DNA. The cross-linked DNA will then be treated with either T7 or SP6 RNA polymerase to produce runoff RNA. Further analysis of the runoff RNA by polyacrylamide gel electrophoresis will enable the investigators to correlate the region cross-linked with the length of RNA formed. Finally, analysis using ligation anchored-PCR on cross-linked DNA excised from the plasmid will more directly reveal where cross-linking occurred on the DNA.