Interactions of two large antiviral polyamides with the long control region of HPV16.
Interactions of two large antiviral polyamides with the long control region of HPV16.
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DOI：
10.1016/j.biochi.2016.04.022
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发表时间：
2016-08
期刊：
影响因子：
3.9
通讯作者：
Dupureur CM
中科院分区：
文献类型：
--
作者：
Vasilieva E;Niederschulte J;Song Y;Harris GD Jr;Koeller KJ;Liao P;Bashkin JK;Dupureur CM
PA1和PA25是大发卡聚酰胺，在细胞培养中几乎可以有效消除HPV16片段（DNA）， PA25对三种致癌型HPV具有广谱活性（Edwards， T. G., Koeller， K. J., Slomczynska， U, Fok， K, Helmus， M., Bashkin， J. K., Fisher， C, Antiviral Res. 91(2011) 177-186）。本文描述了这些PAs与HPV16（7348-122）长控制区（LCR）序列的相互作用。利用PA1的FeEDTA偶联物（设计用于识别5 ' -W2GW7-3 '; W = A或T），检测到该片段的34个亲和切割（AC）模式。这些位点可以用完美、单次、双次、三次和四次错配的序列来合理化。定量dna酶I足迹分析表明，完美位点与PA1结合的Kds在0.7-2.2 nM之间。单、双、三重和四重错配位点的Kds范围为1-3 nM至20 nM。使用AC和EDTA偶联物，我们报告了与较小的8环发夹PAs不同，在这种大聚酰胺中引入手性旋转对结合方向（正向与反向）没有影响。尽管PA25-EDTA偶联物通过两个Im残基识别5 ' -W2GW5GW4-3 '，但在该区域产生了31个亲和切割位点。不含EDTA的PA25的低nM Kds表明对三重和四重错配具有较高的耐受性。虽然研究的序列覆盖范围很广，但两个PAs的AC切割模式显示出离散的结合事件，并且没有明显重叠。这表明，在A/T丰富序列的背景下，这些pa不能识别DNA的一个简单的共享序列相关特征。这些见解继续告知大发夹PA-DNA相互作用和抗病毒行为的复杂性质。
PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5’-W2GW7-3’; W = A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7-2.2 nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3 nM to 20 nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5’-W2GW5GW4-3’ via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicate a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.