Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA.
Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA.
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DOI：
10.1093/nar/gkr860
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发表时间：
2012-02
影响因子：
14.9
通讯作者：
Silverman SK
中科院分区：
文献类型：
--
作者：
Xiao Y;Wehrmann RJ;Ibrahim NA;Silverman SK
我们最近报道了一种DNA催化剂（脱氧核酶）可以在几分钟的时间尺度上定点水解DNA。序列特异性由DNA底物和两个寡核苷酸结合臂之间的Watson-Crick碱基配对提供，所述寡核苷酸结合臂位于脱氧核酶的40-nt催化区的侧翼。来自我们最近的体外选择努力的DNA催化剂10 MD5可以在Zn 2+和Mn 2+辅因子的帮助下切割单链DNA底物序列，只要底物切割位点包含四个特定的核苷酸ATG^T。因此，10 MD5只能切割每256（44）个任意选择的DNA位点中的1个，这是相当差的底物序列耐受性。在这项研究中，我们证明了更广泛的一般性脱氧核酶的位点特异性DNA水解。进行了新的选择实验，揭示了仅向N40 DNA催化区呈递一个或两个未配对的DNA底物核苷酸的最佳性。然后进行全面的选择，在某些情况下包括关键选择压力以在预定位点切割底物。这些努力导致鉴定出许多新的DNA水解脱氧核酶，其中许多在切割位点仅需要两个特定的核苷酸同一性（例如T^G），同时保留沃森-克里克序列的一般性，除了那些核苷酸之外，沿着有用的切割速率。这些研究结果建立了广泛的序列耐受性和位点特异性的脱氧核酶很容易确定的单链DNA的水解实验。
We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn2+ and Mn2+ cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (44) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N40 DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.
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影响因子：
15
作者：
He, Gaofei;Rapireddy, Sirinivas;Ly, Danith H.
通讯作者：
Ly, Danith H.
影响因子：
3.2
作者：
Kost, Diana M.;Gerdt, Joseph P.;Silverman, Scott K.
通讯作者：
Silverman, Scott K.
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：
3.7
作者：
Nickens, David G.;Bardiya, Nirmala;Burke, Donald H.
通讯作者：
Burke, Donald H.