Native Molecular State of Adeno-Associated Viral Vectors Revealed by Single-Molecule Sequencing
Native Molecular State of Adeno-Associated Viral Vectors Revealed by Single-Molecule Sequencing
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DOI：
10.1089/hum.2011.160
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发表时间：
2012-01-01
影响因子：
4.2
通讯作者：
Xiao, Weidong
中科院分区：
文献类型：
--
作者：
Kapranov, Philipp;Chen, Lingxia;Xiao, Weidong
腺相关病毒（AAV）载体的单链基因组是导致缓慢上升但长期的转基因表达动力学的关键因素之一。先前的分子研究已经建立了现在被认为是AAV基因组的教科书分子模型，其在任一末端具有两个反向串联重复拷贝。在这项研究中，我们使用单分子测序（SMS）分析了数十万个直接从衣壳分离的AAV载体DNA分子，避免了任何中间步骤，如质粒克隆。在常规和超大载体的3'端的序列谱确实显示存在反向末端重复序列（ITR），这提供了AAV载体包装从其3'端起始的直接确认。此外，载体5 '-末端谱显示过大载体的不一致终止。这样的不完整载体预期不会经历其基因组DNA的第二链的规范合成，因此只能通过DNA互补链的退火发挥功能。此外，还检测到低水平的污染质粒DNA。SMS可能成为一个有价值的工具，在开发阶段的载体，是候选人的临床使用和促进/加速研究的载体生物学。
The single-stranded genome of adeno-associated viral (AAV) vectors is one of the key factors leading to slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered a textbook molecular model of AAV genomes with two copies of inverted tandem repeats at either end. In this study, we profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from capsids, using single-molecule sequencing (SMS), which avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an inverted terminal repeat (ITR), which provided direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5'-terminus profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.