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hiv药物的毒性和耐药病毒的快速选择。靶向逆转录酶（RT）的RNA适体在细胞内表达时抑制病毒复制，它们提供了小分子药物的潜在替代品。与小分子药物相比，适体被认为更不容易发生突变。该项目的长期目标是确定天然和进化的RT氨基酸序列变化对RT功能和核酸适体抑制的影响。在Aim 1中，我们将克隆，表达和纯化来自多个菌株的RT，重点是HIV-1的主要组和亚型。DNA聚合、RNaseH和链转移活性将在一组抑制RNA适体（含和不含核衣壳NCp7）的挑战中被测量。目的2将定义在病毒攻击人类细胞时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。目的3将确定适体选择过程中的多进化支靶向是否增强了与在选择过程中最初未靶向的进化支的RT的交叉反应性。我们将产生新的RNA适体，抑制与HXB2进化距离增加的成对或组RT。据推测，结合多种RT的适体可以识别高度保守的特征，这些特征较少受到适体抗性突变的影响，这使得它们特别有价值。该项目的结果将在体外建立先前在基于细胞的病毒测定中观察到的对适体挑战的可变敏感性的分子基础，他们将开始测量RT进化逃离抑制的能力，他们将产生更少受逃逸突变影响的新适体。

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.