In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine-pyrimidine junctions.
In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine-pyrimidine junctions.
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DOI：
10.1093/nar/gkn396
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发表时间：
2008-08
影响因子：
14.9
通讯作者：
Li Y
中科院分区：
文献类型：
--
作者：
Schlosser K;Gu J;Lam JC;Li Y
在此，我们寻求基于被称为脱氧核酶的催化DNA分子的新的或改进的核糖核酸内切酶。目前所有的RNA切割脱氧核酶都可以以至少0.1/min的速率切割几乎所有的16种可能的二核苷酸连接，除了嘧啶-嘧啶（pyr-pyr）连接，其切割速度慢1-3个数量级。我们进行了四个单独的体外选择实验以靶向嵌合RNA/DNA底物内的每种pyr-pyr二核苷酸组合（即CC、UC、CT和UT）。我们使用了一个仅包含20个随机序列核苷酸的DNA分子库，以便在每个实验中可以对所有可能的序列排列进行采样。从总共245个克隆中，我们确定了22个不同的序列家族，其中21个代表新的脱氧核酶基序。最快的脱氧核酶对CC、UC、CT和UT接头的kobs值（单次转换，分子间形式）分别为0.12/min、0.04/min、0.13/min和0.15/min。与之前在相同反应条件下报告的最佳速率相比，这些值代表CC和UC接头的6至8倍改善，以及CT和UT接头的1000至1600倍改善。相同的脱氧核酶对所有RNA底物的活性都低1000倍，但通过进一步的体外进化和工程改造可能会得到改善。
Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited kobs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ∼1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.
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影响因子：
15
作者：
Guckian, KM;Schweitzer, BA;Kool, ET
通讯作者：
Kool, ET
DOI：
10.1073/pnas.94.9.4262
发表时间：
1997-04-29
影响因子：
11.1
作者：
Santoro, SW;Joyce, GF
通讯作者：
Joyce, GF
影响因子：
15
作者：
Li, YF;Breaker, RR
通讯作者：
Breaker, RR
影响因子：
46.9
作者：
Cairns, MJ;Hopkins, TM;Sun, LQ
通讯作者：
Sun, LQ
影响因子：
15.9
作者：
Khachigian, LM
通讯作者：
Khachigian, LM