We want to understand several basic biochemical problems primarily related to various gene controk regions and how they function. Our approach to this problem is somewhat unique and involves manipulating and modifying polynucleotide sequences (usually obtained by chemical and enzymatic synthesis procedures) in a defined manner and then observing how these changes alter the function of a control region. In order to complete this objective, we need a sound, basic research oriented subgroup primarily interested in developing the chemistry of polynucleotides. This grant application is to provide funds for that purpose.
The specific aims of this research are to improve and further develop new procedures for synthesizing polynucleotides on a polymer support and to develop and test new nucleoside and phosphorus analogs useful for studying protein-DNA interactions. Funds previously provided from this grant were used to develop a new, far superior methodology for synthesizing deoxyoligonucleotides. We can now rapidly synthesize micromole quantities of deoxyoligonucleotides containing 10 to 15 mononucleotides. These compounds are synthesized on a silica gel support and isolated in pure form by a simple, one step procedure. We now propose to expand and further develop this procedure which utilizes nucleoside phosphites as activated intermediates. This is a relatively new class of compounds only superfically investigated at this time. We propose to survey these intermediates more completely and consequently define their limitations and advantages. Additionally we want to investigate other silica gels and other support matrices. The objective of these projects is to develop procedures for the rapid, chemical synthesis of large gene control regions (100-200 base pairs) in modified and unmodified form. The phosphite chemistry is ideally adapted to synthesis of polynucleotides containing a large variety of base and phosphorus analogs. We propose to develop a new class of such analogs. These analogs would be found within deoxyoligonucleotides at specific, predefined positions and selected for synthesis based upon how they might alter protein-DNA interaction sites. We propose to use these analogs to probe protein-DNA interactions in gene control regions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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University of Colorado at Boulder
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United States
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