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首页> 外文期刊>Nucleic Acid Therapeutics >DNA Aptamers to Human Immunodeficiency Virus Reverse Transcriptase Selected by a Primer-Free SELEX Method: Characterization and Comparison with Other Aptamers
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DNA Aptamers to Human Immunodeficiency Virus Reverse Transcriptase Selected by a Primer-Free SELEX Method: Characterization and Comparison with Other Aptamers

机译：DNA适体对人免疫缺陷病毒逆转录酶的逆转录酶选择由底漆的SELEX方法选择：表征和与其他适体的比较

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摘要

A 30-nucleotide DNA aptamer (5'-AGGAAGGCTTTAGGTCTGAGATCTCGGAAT-3', denoted PF1) selected for high affinity to human immunodeficiency virus reverse transcriptase (HIV RT) using a primer-free SELEX (systematic evolution of ligands by exponential enrichment) method was characterized to determine features promoting tight binding. PF1's equilibrium dissociation constant for RT was similar to 80 nM, over 10-fold lower than a random 30-mer. Changing the 2 terminal diguanosine repeats (underlined above) to diadenosine or dithymidine modestly decreased binding. Any changes to the 2 central diguanosines dramatically decreased binding. Binding was highly sensitive to length, with any truncations that deleted part of the 4 diguanosine motifs resulting in a 6-fold or more decrease in affinity. Even a construct with all the diguanosine motifs but lacking the 5' terminal A and 3 nucleotides at the 3' end showed similar to 3-fold binding decrease. Changes to the nucleotides between the diguanosines, even those that did not alter PF1's low secondary structure (free energy of folding Delta G = -0.61 kcal/mol), dramatically decreased binding, suggesting sequence specificity. Despite the diguanosine motifs, circular dichroism (CD) spectra indicated that PF1 did not form a G-quartet. PF1 inhibited HIV RT synthesis with a half-maximal inhibitory value (IC50) of similar to 60 nM. Larger, more structured RT DNA aptamers based on the HIV polypurine tract and those that formed G-quartets (denoted S4 and R1T) were more potent inhibitors, with IC50 values of similar to 4 and similar to 1 nM, respectively. An RNA pseudoknot aptamer (denoted 1.1) showed an IC50 near 4 nM. Competition binding assays with PF1 and several previously characterized RT aptamers indicated that they all bound at or near the primer-template pocket. These other more structured and typically larger aptamers bound more tightly than PF1 to RT based on filter binding assays. Results indicate that PF1 represents a new class of RT aptamers that are relatively small and have very low secondary structure, attributes that could be advantageous for further development as HIV inhibitors.
机译：一种30核苷酸DNA适体（5'-AggaAGGCTTAGGTCTGAGATCTCGGAT-3'，表示的PF1），用于使用无引物切片（通过指数富集的配体的系统演化）方法对人免疫缺陷病毒逆转录酶（HIV RT）进行高亲和力。确定促进紧张绑定的特征。用于室温的PF1平衡解离常数与80nm相似，比随机30-mer低10倍。改变2末端的偶昆氨酸骨（上面带下划线），以二角一碱或Dithymidine适度降低的结合。 2中央沸点的任何变化显着降低了绑定。结合对长度高度敏感，具有截断的任何截短，其中4个偶像氨酸基序的一部分导致6倍或更低的亲和力降低。甚至在3'末端缺少5'末端A和3个核苷酸的含有所有达胍基序的构建体也类似于3倍的结合减少。对偶胍之间的核苷酸的变化，即使那些没有改变PF1的低二级结构的那些（折叠δG= -0.61kcal / mol的自由能），均显着降低，表明序列特异性。尽管达川源素图案，圆形二色性（CD）光谱表明PF1没有形成G-Quartet。 PF1抑制HIV RT合成，其半最大抑制值（IC 50）类似于60nm。基于HIV多嘌呤道的较大，更多的结构RT DNA适体和形成G-四（表示的S4和R1T）的那些更有效的抑制剂，其IC50分别与4和类似的1nm。 RNA pseudoknot适体（表示为1.1）显示IC50接近4nm。具有PF1的竞争结合测定和几个先前表征的RT适体表明它们在底漆模板口袋处或附近界定。这些其他结构和通常更大的适体基于过滤器结合测定基于过滤器结合测定粘合比PF1至室温更紧密。结果表明，PF1代表了一种新的RT适体，其相对较小，具有非常低的二级结构，其可能是有利的，用于进一步发展为HIV抑制剂。

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  • 来源
    《Nucleic Acid Therapeutics》 |2012年第3期|共15页
  • 作者

    Lai Yi-Tak; DeStefano Jeffrey J.;

  • 作者单位

    Department of Cell Biology and Molecular Genetics University of Maryland College Park College Park Maryland 20742 USA;

    Department of Cell Biology and Molecular Genetics University of Maryland College Park College Park Maryland 20742 USA;

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  • 正文语种 eng
  • 中图分类 生物化学;
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