Lauhon C T, Szostak J W
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
J Am Chem Soc. 1995 Feb 1;117(4):1246-57. doi: 10.1021/ja00109a008.
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 (Kd = 1-5 micromolar). 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.
通过体外筛选已分离出特异性结合核黄素（Rb）和β-烟酰胺单核苷酸（NMN）的RNA分子。发现一种包含分子内G-四联体的简单结构基序能紧密结合氧化型核黄素（解离常数Kd = 1 - 5微摩尔）。共有序列的DNA版本也能结合，但亲和力较弱。二甲基亚砜（DMS）保护实验表明，这些适体的四联体结构通过与黄素相互作用而稳定。作为其氧化还原特异性的一种衡量，这些适体在一种与核黄素结构相近的5-脱氮核黄素衍生物的氧化型和还原型之间未表现出显著的差异结合。与核黄素适体缺乏氧化还原特异性相反，筛选出的用于结合NAD中烟酰胺部分的RNA在溶液中能区分NAD和NADH，差异超过一个数量级。基于其中一个NMN适体序列构建的诱变文库用于重新筛选NMN结合。对重新筛选的序列进行比较，从而确定了适体的结合区域。对于最小的NMN结合RNA，提出了一种包含两个相互作用茎环的复杂二级结构。使用同一个诱变文库来筛选增强对NMN和NMNH区分能力的RNA。从这些重新筛选的序列中，鉴定出结合区域内的一个突变，该突变增加了对NMN的特异性。这些实验表明，RNA能以低微摩尔亲和力结合这些辅因子，并且就烟酰胺辅因子而言，能区分两种氧化还原状态。这些辅因子结合基序可能为生成新的核酶提供一个框架，这些核酶可催化类似于基本代谢途径中发现的氧化还原反应。
