DETERMINATION OF SEQUENCE & IDENTITY OF HIV MRNA & SRP RNA FRAGMENTS

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Our lab has been engaged in the structure determination of RNA and DNA fragments using nuclear magnetic resonance and computational modelling tools for quite some years. Recent RNA projects involve the determination of the solution structure of a conserved RNA sequence in the LTR of HIV-1 shortly downstream from the TAR sequence. The 21 mer RNA has been chemically synthesized and the NMR studies were not fully completed when we realized that the material was slowly decomposing. Another large portion of RNA was synthesized that however gave significantly different NMR spectra. We need to characterize old and new materials to complete our structural work. Without this information we would have to spend several months to re-acquire many 2D NMR sets. Therefore we propose to use mass spec to analyze sequence and identity of the different batches of RNA. In another project, we are studying the structure of a conserved motif of SRP RNA (domain IV, 43 nucleotides). RNAs of that size are synthesized via in vitro transcription. This enzymatic process often leaves us with a mixture of products including the full length transcript and most commonly a species with an additional 3'-residue. This additional longer RNA fragment is difficult to remove and causes problems in the NMR assignment process. Since the RNA transcripts are PAGE purified, one cannot necessarily make assumptions about which of the spatially very tightly packed bands on the gel represents the full length product. Mass spec would not only help us identify which of the bands is the desired product but also would give us an idea if the extra 3'-nucleotide is a specific one or a mixture. This information would help the NMR assignment process enormously since we would know what signals to expect.