RNA aptamers to inhibit natural and evolved RT variants

描述(由申请人提供)：需要新的治疗策略来规避当前抗艾滋病毒药物的毒性和快速选择耐药病毒。针对逆转录酶(RT)的RNA适配子在细胞内表达时抑制病毒复制，它们提供了一种潜在的小分子药物的替代品。与小分子药物相比，适体被认为是不太容易发生逃逸突变的。该项目的长期目标是确定RT氨基酸序列变异--无论是自然的还是进化的--对RT功能和核酸适配子抑制的影响。在目标1中，我们将从几个毒株中克隆、表达和纯化RT，重点介绍HIV-1的主要群组和亚型。在一组抑制性RNA适配子的挑战中，无论有没有核衣壳NCp7，都会测量DNA聚合、RNAseH和链转移活性。目的2将定义在人类细胞的病毒挑战中赋予适体抗性的氨基酸，并将确定体外生化参数是否与细胞内行为相关。这一目标构成了我们研究计划的一项重大新举措。目标3将确定适体选择期间的多分支靶向是否增强了与来自选择期间最初未靶向的分支的RT的交叉反应。我们将产生新的RNA适配子，在与HXB2的进化距离不断增加的情况下抑制RT对或RT集。结合不同RT的适体被假设为识别高度保守的特征，这些特征不太容易受到适体耐药性突变的影响，从而使它们特别有价值。该项目的结果将在体外建立对以前在基于细胞的病毒检测中观察到的适体挑战的可变敏感性的分子基础，它们将开始测量RT的进化逃逸抑制的能力，并且它们将产生不太容易逃逸突变的新的适体。

DESCRIPTION (provided by applicant): New therapeutic strategies are needed to circumvent the toxicity of current anti-HIV drugs and the rapid selection of drug-resistant virus. RNA aptamers targeted to the reverse transcriptase (RT) inhibit viral replication when expressed inside cells, and they offer a potential alternative to small molecule drugs. Aptamers are hypothesized to be less susceptible escape mutations than small molecule drugs. The long-term goal of this project is to define the influence of RT amino acid sequence variations-both natural and evolved-on RT function and on inhibition by nucleic acid aptamers. In Aim 1, we will clone, express and purify RT from several strains, emphasizing the major HIV-1 groups and subtypes. DNA polymerization, RNaseH and strand transfer activities will be measured for each RT in challenges from a panel of inhibitory RNA aptamers, with and without nucleocapsid NCp7. Aim 2 will define the amino acids that confer aptamer resistance in viral challenges of human cells, and will establish whether in vitro biochemical parameters correlate with intracellular behavior. This aim constitutes a major new initiative for our research program. Aim 3 will determine whether multi-clade targeting during aptamer selection enhances cross-reactivity with RT from clades that were not initially targeted during the selection. We will generate new RNA aptamers that inhibit pairs or sets of RT at increasing evolutionary distances from HXB2. Aptamers that bind diverse RT are hypothesized to recognize highly conserved features that are less subject to aptamer resistance mutations, making them especially valuable. The results from this project will establish in vitro the molecular basis for variable sensitivities to aptamer challenges previously observed in cell-based viral assays, they will begin to measure RT's capacity to evolve escape from inhibition, and they will generate new aptamers that are less subject to escape mutations.