NMR DETERMINATION OF POXVIRUS DNA BULGED STRUCTURE

我们正在确定两个天然分子的高分辨结构 出现包含螺旋外碱基的痘苗病毒DNA片段 核磁共振。每个片段都包含两个额外的碱基(G和C 链，由d(Aa).d(Tt)分隔。序列出现在牛痘病毒中 基因组作为反向互补，每个末端附近一个，其中 位于相应但互补位置的螺旋外碱基。我们的 策略是合成两个29mer： 5‘CCTAATTATAACGAAGTTAGTACATTAGG3’和 5‘CCTAATGTACTAACGAAGTTATAATTAGG3’。我们还在合成和 确定缺少任何一种的对照寡核苷酸的结构 或者是两个螺旋外的碱基。这两个29mer含有病毒 DNA序列，末端d(AT)碱基对已被替换 由d(GC)对和三核苷酸连接物GAA添加到 允许形成阀杆/环。这将减少 NOESY信号中的磨损以及重叠，避免了必要性 通过混合单股形成双链，并提高整体 稳定性。我们将用以下方法确定DNA茎/环结构 受限分子动力学，使用Mardigras，基于两者 NOE导出的质子-质子距离和弛豫矩阵--计算 NOESY在D2O和H2O中的交叉峰体积。MidasPlus和Sparky 是我们评估结构性特征工作的核心，例如 两个AT碱基对在外螺旋之间的配对程度 碱基与螺外碱基的环化或堆积 他们自己。

We are determining the high resolution structures of two naturally occurring vaccinia virus DNA segments containing extrahelical bases by NMR. Each segment contains two extra bases (G and C) on the same strand, separated by d(AA).d(TT). The sequences appear in the vaccinia genome as reverse complements, one near each terminus, with the extrahelical bases at corresponding but complementary locations. Our strategy is to synthesize two 29mers: 5'CCTAATTATAACGAAGTTAGTACATTAGG3' and 5'CCTAATGTACTAACGAAGTTATAATTAGG3'. We are also synthesizing and determining the structures of control oligonucleotides lacking either or both of the extrahelical bases. The two 29mers contain the viral DNA sequences, with the terminal d(AT) base pairs having been replaced by d(GC) pairs and a trinucleotide linker GAA having been added to permit formation of a stem/loop. This will reduce the effects of fraying as well as overlap in the NOESY signals, avoid the necessity of forming a duplex by mixing single strands, and improve overall stability. We will determine the DNA stem/loop structures with restrained molecular dynamics, using MARDIGRAS, based upon both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes in both D2O and H2O. MidasPlus and Sparky are central to our work in assessing structural features such as the extent of pairing of the two AT base pairs between the extrahelical bases and the looping out or stacking of the extrahelical bases themselves.