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

本实验室一直从事RNA的结构测定， 使用核磁共振和计算的DNA片段 多年来的模型工具。 最近的RNA项目涉及 确定一个保守的RNA序列的溶液结构， HIV-1的LTR位于TAR序列的下游。 21 Mer RNA是化学合成的，核磁共振研究还不完全。 当我们意识到材料正在慢慢分解时，我们完成了。 另一大部分RNA是合成的， 明显不同的NMR谱。 我们需要描述旧的， 新材料来完成我们的结构工作。 没有这个 我们将不得不花几个月的时间重新获得许多信息， 2D NMR集。 因此，我们建议使用质谱分析 不同批次RNA的序列和身份。 在另一 我们正在研究SRPRNA的保守基序的结构， （结构域IV，43个核苷酸）。 这种大小的RNA是通过 体外转录 这种酶促过程通常会给我们留下 包括全长转录物和大多数转录物的产物的混合物 通常是具有额外的3 '-残基的种类。 该附加 较长的RNA片段很难去除，并导致 NMR分配过程。 由于RNA转录物是PAGE纯化的， 我们不一定能假设 凝胶上非常紧密的条带代表了全长 产品 质谱不仅能帮助我们确定 是我们想要的产品，但也会给我们一个想法，如果额外的 3 ′-核苷酸是特定的一种或混合物。 这一信息将 极大地帮助了核磁共振分配过程，因为我们会知道 信号期待

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.