NMR DETERMINATION OF POXVIRUS DNA BULGED STRUCTURE

我们正在确定两个天然的高分辨率结构， 含有螺旋外碱基的牛痘病毒DNA片段， 匪r 每个片段包含两个额外的碱基（G和C）， 链，由d（AA）.d（TT）分开。序列出现在牛痘中 基因组作为反向互补，每个末端附近一个， 在相应但互补的位置的extrahelical基地。 我们 策略是合成两个29聚体： 5 'CCTAATTATAACGAAGTTAGTACATTAGG 3'和 5'CCTAATGTACTAACGAAGTTATAATTAGG3'. 我们也在合成， 确定缺少以下任一者的对照寡核苷酸的结构 或两个螺旋外基部。 两个29聚体含有病毒 末端d（AT）碱基对被替换的DNA序列 通过将d（GC）对和三核苷酸接头GAA添加到 允许形成茎/环。 这将减少影响， NOESY信号中的磨损和重叠，避免了 通过混合单链形成双链体， 稳定 我们将确定DNA茎/环结构， 限制分子动力学，使用MARDIGRAS，基于两者 NOE导出的质子-质子距离和弛豫矩阵-计算 D2 O和H2O的NOESY交叉峰体积。 MidasPlus和Sparky 是我们评估结构特征工作的核心， 外螺旋之间两个AT碱基对的配对程度 碱基和螺旋外碱基的成环或堆叠 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 The two 29mers contain the viral DNA sequences, with the terminal d(AT) base pairs having been replaced by d(GC) pairs and a trinucleotide linker GAA having been added to permit formation of a stem/loop.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.