Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydrolyzing deoxyribozyme.
Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydrolyzing deoxyribozyme.
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DOI：
10.1021/bi1013672
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发表时间：
2010-11-09
期刊：
影响因子：
2.9
通讯作者：
Silverman, Scott K.
中科院分区：
文献类型：
--
作者：
Xiao, Ying;Chandra, Madhavaiah;Silverman, Scott K.
我们最近报道了10 MD5的体外筛选鉴定，10 MD5是一种需要Mn ~（2+）和Zn ~（2+）来水解单链DNA底物并形成5′-磷酸和3′-羟基末端的脱氧核酶。10 MD5的DNA切割进行，kobs = 2.7 h-1，与未催化的P-O水解反应相比，速率提高1012。10 MD5具有接近7.5的非常尖锐的最适pH，当pH在任一方向上仅改变0.1单位时，DNA切割速率和产率大大降低。在这里，我们通过重新选择（体外进化）优化了10 MD5，导致具有更宽pH耐受性的变体，这对实际DNA切割应用很重要。由于脱氧核酶和底物之间广泛的Watson-Crick互补性，亲本10 MD5固有地具有序列特异性;即，它能够优先于其它序列切割一种DNA底物序列。10 MD5也是位点特异性的，因为DNA底物内只有一个磷酸二酯键被切割，尽管在这里我们表明，故意在切割位点附近产生沃森-克里克错配放松了位点特异性。新进化的10 MD5变体如9 NL 27也是序列特异性的。然而，9 NL 27位点特异性对于一些底物序列是松弛的，即使当完全沃森-克里克互补性被维持时，对应于pH耐受性和位点特异性之间的功能折衷。9 NL 27的位点特异性可以通过将其“识别位点”从ATG 4 T（如对于10 MD5）扩展到ATG 4 TT或更大来恢复;即，通过认为9 NL 27相对于10 MD 5具有降低的底物序列耐受性。这些发现提供了基本的见解之间的相互作用的关键脱氧核酶的耐受性和选择性的特点，与实际的DNA催化的DNA水解的持续发展的影响。
We recently reported the identification by in vitro selection of 10MD5, a deoxyribozyme that requires both Mn2+ and Zn2+ to hydrolyze a single-stranded DNA substrate with formation of 5′-phosphate and 3′-hydroxyl termini. DNA cleavage by 10MD5 proceeds with kobs = 2.7 h−1 and rate enhancement of 1012 over the uncatalyzed P–O hydrolysis reaction. 10MD5 has a very sharp pH optimum near 7.5, with greatly reduced DNA cleavage rate and yield when the pH is changed by only 0.1 unit in either direction. Here we have optimized 10MD5 by reselection (in vitro evolution), leading to variants with broader pH tolerance, which is important for practical DNA cleavage applications. Because of the extensive Watson-Crick complementarity between deoxyribozyme and substrate, the parent 10MD5 is inherently sequence-specific; i.e., it is able to cleave one DNA substrate sequence in preference to other sequences. 10MD5 is also site-specific because only one phosphodiester bond within the DNA substrate is cleaved, although here we show that intentionally creating Watson-Crick mismatches near the cleavage site relaxes the site specificity. Newly evolved 10MD5 variants such as 9NL27 are also sequence-specific. However, the 9NL27 site specificity is relaxed for some substrate sequences even when full Watson-Crick complementarity is maintained, corresponding to a functional compromise between pH tolerance and site specificity. The site specificity of 9NL27 may be restored by expanding its “recognition site” from ATG^T (as for 10MD5) to ATG^TT or larger; i.e., by considering 9NL27 to have reduced substrate sequence tolerance relative to 10MD5. These findings provide fundamental insights into the interplay among key deoxyribozyme characteristics of tolerance and selectivity, with implications for ongoing development of practical DNA-catalyzed DNA hydrolysis.
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影响因子：
3.2
作者：
Kost, Diana M.;Gerdt, Joseph P.;Silverman, Scott K.
通讯作者：
Silverman, Scott K.
影响因子：
15
作者：
Flynn-Charlebois, A;Wang, YM;Silverman, SK
通讯作者：
Silverman, SK
影响因子：
16.6
作者：
Silverman, Scott K.
通讯作者：
Silverman, Scott K.
DOI：
10.1073/pnas.0510879103
发表时间：
2006-03-14
影响因子：
11.1
作者：
Schroeder, GK;Lad, C;Wolfenden, R
通讯作者：
Wolfenden, R
影响因子：
15
作者：
Flynn-Charlebois, A;Prior, TK;Silverman, SK
通讯作者：
Silverman, SK