STRUCTURE AND FUNCTION OF NUCLEIC ACID THERAPEUTICS

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The objective of this research is a detailed investigation of the structure and function of nucleic acid analogs. Using X-ray crystallography, the three-dimensional structures of chemically modified nucleic acid fragments will be determined. The first focus of the project is to use the accumulated structural data and subsequent structure-stability and structure-activity correlations as guides for designing the next generation of antisense therapeutics with potential anticancer, antiviral and antiinflammatory indications. Improvements based on the structural insights of the following key features of antisense compounds will be given particular attention: RNA affinity, nuclease resistance and susceptibility of the antisense-RNA hybrid to RNase H-mediated cleavage. The efficacy of the newly designed modifications will be tested in collaboration with Isis Pharmaceuticals Inc., using a variety of in vitro and cell-based assays. In addition to their potential use as therapeutic antisense reagents, nucleic acid analogs are a prerequisite for studying the origin and evolution of natural DNA and RNA. Their altered properties relative to the latter make them ideal tools in medical diagnostics, material science, analysis of protein-nucleic acid interactions, DNA electron transfer etc. The second. focus of this project is the structure determination and structure-function analysis of several nucleic acid analogs that are being explored in some of the above areas. Moreover, the favorable crystallization properties observed with certain analogs will be exploited for studying DNA-ion interactions at ultra-high resolutions. The specific aims of this research are: 1) Analysis of the structural origins of the RNA affinity of antisense nucleic acid analogs. 2) Analysis of the structural origins of the nuclease resistance of antisense nucleic acid analogs. 3) Structure-guided design of third- generation antisense modifications using principles emerging from studies l and 2. Evaluation of the properties of antisense oligonucleotides containing these modifications, using UV melting experiments, enzyme assays and cell-based assays. 4) Investigation of the structural origins of the substrate specificity of E. coli RNase H by way of structure analysis of hybrids between RNA and conformationally restricted analogs that are processed by the enzyme. 5) X-ray crystallographic analysis of the structures of artificial nucleic acid pairing systems, e.g. DNAs with hexose-based sugars or stilbene caps. A rationalization of the thermodynamic stabilities, pairing properties and particular functional aspects of the individual analogs based on structural data. 6) Determination of crystal structures of DNAs and chemically modified DNAs at ultrahigh resolutions to analyze metal ion coordination to DNA. Based on these and their comparisons with reference structures of similar precision, a detailed analysis of the role of alkali and earth alkali metal ions in DNA structure and packing. 7) Development of data collection and structure determination protocols for nucleic acid crystals that exploit the anomalous scattering component of selected alkali and earth alkali metal ions, sulfur (in analogs) and phosphorus.