RNA APTAMERS THAT BIND FLAVIN AND NICOTINAMIDE REDOX COFACTORS
RNA APTAMERS THAT BIND FLAVIN AND NICOTINAMIDE REDOX COFACTORS
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DOI：
10.1021/ja00109a008
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发表时间：
1995-02-01
影响因子：
15
通讯作者：
SZOSTAK, JW
中科院分区：
文献类型：
--
作者：
LAUHON, CT;SZOSTAK, JW
通过体外筛选分离出了特异性结合核黄素（Rb）和β-烟酰胺单核苷酸（NMN）的RNA分子。发现含有分子内G-四联体的简单结构基序与氧化核黄素（Kd = 1-5 μ M）紧密结合。DMS保护实验表明，这些适体的四重结构通过与黄素的相互作用而稳定。作为其氧化还原特异性的量度，适体在作为核黄素的接近结构类似物的5-脱氮核黄素衍生物的氧化和还原形式之间不显示显著的差异结合。与核黄素适体缺乏氧化还原特异性相反，选择用于结合NAD的烟酰胺部分的RNA在溶液中区分NAD和NADH超过一个数量级。使用基于NMN适体序列之一的诱变池重新选择NMN结合。重新选择的序列的比较导致适体的结合区域的鉴定。一个复杂的二级结构，包含两个相互作用的茎环提出了最小的NMN结合RNA。使用相同的诱变合并液来选择NMN和NMNH之间增加的区分度。从这些重新选择的序列中，鉴定了结合区内的突变，其增加了对NMN的特异性。这些实验表明，RNA可以结合这些辅因子与低微摩尔亲和力，并在烟酰胺辅因子的情况下，可以区分两种氧化还原状态。这些辅因子结合基序可以提供一个框架，产生新的核酶，催化氧化还原反应类似于那些发现在基本的代谢途径。
RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection, A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (K-d = 1-5 mu M) DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.