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

我们实验室一直致力于核糖核酸的结构测定和 利用核磁共振和计算技术获得DNA片段 建模工具已经有好几年的历史了。最近的RNA项目涉及 一种保守的RNA序列溶液结构的测定 在TAR序列下游不久的HIV-1的LTR。21世纪末 核糖核酸是化学合成的，核磁共振研究还没有完全完成。 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被合成，然而给出了 明显不同的核磁共振波谱。我们需要把旧的和 新材料来完成我们的结构工作。如果没有这个 我们将不得不花几个月的时间来重新获得许多 2D核磁共振组。因此，我们建议使用质谱仪来分析 不同批次的RNA的序列和鉴定。在另一个国家 项目中，我们正在研究SRP RNA的一个保守基序的结构 (结构域IV，43个核苷酸)。这种大小的RNA是通过In合成的 体外转录。这种酶促过程通常会给我们留下一个 混合产品，包括完整的成绩单和MOST 通常是具有额外的3-残基的物种。这是额外的 较长的RNA片段很难移除，并在 核磁共振指认过程。由于RNA转录本是PAGE纯化的， 人们不一定能假设哪一种空间上的 凝胶上紧密堆积的条带代表全长 产品。质谱仪不仅能帮助我们识别哪些波段 是想要的产品，但也会给我们一个想法，如果额外的 3‘-核苷酸是一种特定的核苷酸或其混合物。这些信息将会 极大地帮助了核磁共振分配过程，因为我们将知道 期待的信号。

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.