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基于ssDNA的晶体结构抑制剂的基本原理设计结合到APOBEC

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图库
(a) Ribbon schematic of A3A-ssDNA complex showing flipped-out target C and -1 T nucleotides; overall U-shaped binding conformation. (b) Molecular surface of A3A active site; surrounding loops color-coded, superposition of stick-models of ssDNA bound to 4 different molecules in crystal’s asymmetric unit. (c) Representative ssDNA molecule shown; nucleobases and key amino side chains from active site loops labeled. (d) Wall-eye stereo view of A3A active site; bound ssDNA molecule shown in sticks, hydrogen bonds indicated by yellow dashed lines.(a) Ribbon schematic of ssDNA-A3Bctd* complex showing the flipped-out target C (0) and -1 T, as well as the overall U-shaped binding conformation. (b) A superposition of the active site region of A3A (cyan) and A3Bctd* (magenta) with relevant ssDNA substrates (opaque from a representative A3A structure and yellow from the A3Bctd* structure) showing the near-identical positioning of the flipped-out target C and -1 T. (c-d) Composite omit 2Fo-Fc map contoured at 1.0 shown for region surrounding the target cytosine (panel c) or -1 thymine (panel d). (e) Deaminase activity of wild-type A3A on ssDNA substrates containing normal T or the indicated analogs at the -1 position demonstrating that the 5-methyl group is unconstrained structurally. Uracil DNA glycosylase (UDG) readily excises dU and 5FdU from ssDNA and accounts for the 11-nucleotide product in the absence of deamination. However, due to A3A activity on the target C and the 3'-end label, only the shorter 10-nucleotide product is apparent upon deamination and gel fractionation. (f) A wall-eye stereo view of the A3Bctd* active site and the bound ssDNA molecule shown in sticks. Hydrogen bonds are indicated by yellow dashed lines. Water molecules are represented by small red crosshairs.
类别
研究人员
鲁本·哈里斯博士
教授,生物化学系,分子生物学,生物物理学和
外部链接 (harris.cbs.umn.edu)
相原秀树博士
副教授,生物化学系,分子生物学,生物物理学和
外部链接 (cbs.umn.edu)
由...管理
凯文镍
技术授权官 612-625-7289
专利保护

PCT专利申请us20180170984a1
出版物
有针对性的DNA胞嘧啶脱氨和诱变通过APOBEC3A和APOBEC3B结构基础
Nature Structural & Molecular Biology , 体积24,131-139页(2017)
在活细胞中的荧光记者进行定量分析,DNA编辑富集APOBEC-cas9或裂解通过cas9
核酸研究, 第46卷,第14期,2018年8月21日,页E84

DNA碱基编辑以高特异性和效率的酶

新方法将有助于通过修改cas9-APOBEC融合多肽实现用更少的脱靶问题的具体DNA编辑事件。可用于抑制剂的合理设计为单链DNA结合的APOBEC3A和APOBEC3B晶体结构阻止病毒和肿瘤中的可进化性和APOBEC介导的基编辑记者(琥珀色)系统的开发。

体内DNA编辑更好的工具

Better tools are needed for DNA editing in vivo. The original APOBEC1-Cas9 base editing complexes have wide “editing windows”, which are nearly as big as the >20 nucleotide single-stranded DNA region displaced by annealing of the guide-RNA that directs the editing complex. The high-resolution structural information for APOBEC3A and APOBEC3B (catalytic domain) enzymes in complex with relevant single-stranded DNA substrates provide atomic-level explanations for their strong specificity for 5’-TC-3’ dinucleotide sequences within longer single-stranded DNA substrates. They also provide strong structural rationale that has already enabled the local specificity of these enzymes to be changed to 5’-CC-3’. Structural information for these extremely efficient enzymes makes it possible to tune the enzyme to preferentially edit 5’-AC-3’ and 5’-GC-3’ dinucleotide targets. Thus, these enzymes expand the DNA editing toolkit to be able to selectively target DNA cytosine bases in any dinucleotide context. In addition, unlike the normal CRISPR system, base editing requires neither double-strand DNA cleavage nor a DNA donor template.

互补技术

apobec3a-和APOBEC3B(催化结构域)-cas9基编辑络合物可以与适当的向导RNA几乎任何DNA靶进行定位。它们也可结合使用荧光报告用于DNA编辑(即,apobec-和cas9介导的编辑(ACE)报告系统)。

发展阶段

  • 概念验证。进一步细化和优化去。

好处

  • 实现用较少的脱靶问题的具体DNA编辑事件
  • 使编辑复合物的另外的微调由结构引导的诱变和/或高通量筛选
  • 使单链DNA编辑为化学改性剂屏幕(抑制剂或激活剂)

特征

  • 依赖于高分辨率晶体结构信息的ssDNA结合APOBEC3A和APOBEC3B
  • 可以与用于DNA编辑荧光报告(ACE)单独或组合使用

应用

  • 在活细胞中的基因编辑
  • 抑制剂的合理设计来阻止病毒和肿瘤的演化性
  • 与DNA编辑荧光报告(即,apobec-和cas9介导的编辑(ACE)报告系统)单独或组合使用


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