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Synthesis of non-uniformly isotope labelled - "nmr-window" - biologically functional dna and rna and their conformational study by high-field nmr

Objective



NMR spectroscopy is one of the most useful approaches to understand how the structure and dynamics of DNA or RNA an their interactions with various ligands dictate their specific biological functions under closely physiological condition. It It been demonstrated in Prof. Chattopadhyaya's lab (i.e. the host lab) that the use of non-uniform but specific deuterium labelled large DNA and RNA solve the resonance overcrowding problem of IH-NMR spectra of natural DNA and RNA (Tetrahedron) 48, 9033 (1992); ibid, 51, 100065 (1995)). The host lab has also developed large scale synthetic methodologies for a deuterated nucleotide blocks for the automated solid-phase synthesis in order to introduce the NMR-window in large oligo- RNA and DNA, in conjunction with natural ones. The first proof of this NMR-window concept has been unambiguous demonstrated through the synthesis and NMR structure determination of a 20-mer self-complementary DNA duplex (Nucleic acid Res., 21, 5005 (1993)), a 21 mer RNA hairpin loop (Nucleic acid Res., 24 1187 (1996)) and a 31-mer RNA subunit of RNase P RNA (Nucleic acid Res., 24, 2022 (1996)). These findings clearly show that the NMR- window concept indeed simplifies the NMR spectra enormously for the NMR structure determination of large biologically-ffinctional DNA and RN molecules. We herein propose to devise appropriate synthetic routes for triple-labeled oligo-DNA and -RNA in which 13C and 15N labels will be included in the NMR- window part, and the regions outside the NMR- window will be fully-deuterated to afford the non-uniformly isotope labelled biologically functional DNA and RNA. These triple-labeled oligo-DNA/RNA and their complexes with various ligands are ideally suited for determination of their tertiary structures and dynamics by triple resonance NMR which in turn would provide a much improved understanding of the structure, dynamics and activity relationship for large biologically functional DNA/RNA, which has been hithertofore impossible. The full realization of this non-uniform isotope labelling strategy for large oligo-DNA or RNA can only be achieved with the development of new chemistry, both at the monomeric and large biologically functional oligomeric level, and evaluating them by state-of-the-a triple resonance spectroscopy. In this endeavour, the synthesis of non-uniformly deuterium and 13C/l5N labelled catalytic hammerhead RNA is proposed. The NMR structure determination of this hammerhead RNA, both in the presence and absence of Mg2+ for the first time will reveal the dynamic aspects of mechanistic details of RNA catalysis, in general. This knowledge will in turn give insight for the design of appropriate RNA enzyme mimics as therapeutic agents against pathogen and tumor. This project encompasses the goal designated under the ' Structural Biology' section of the Biotechnology program of EU. Training content (objective, benefit and expected impact) The objective is to develop synthetic methodologies of large oligo-DNA and RNA in which l3C and l5N labels have been specifically introduced in the
these triple labelled DNA/RNA by means of multinuclear NMR techniques. After this training, I intend to go back to Leuven to carry with my research objectives on this structural and functional aspects of biopolymers using the tools of both chemistry and NM spectroscopy, either at an industrial or academic capacity. With this acquired knowledge I will be able to start a new research group in the field of Bioorganic Chemistry. Links with industry / industrial relevance (22)

Funding Scheme

RGI - Research grants (individual fellowships)

Coordinator

Uppsala University
Address
Husargatan 3
753 31 Uppsala
Sweden