Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.
J Am Chem Soc. 2023 Apr 5;145(13):7504-7515. doi: 10.1021/jacs.3c00612. Epub 2023 Mar 24.
The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model, the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extensions on a subset of kinetically trapped partially base-paired configurations, followed by the shuffling of these configurations to enable continued oligonucleotide elongation. Here, we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of lengths 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides and investigated the effect of factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high-temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is preactivated. We discuss the necessity of ongoing in situ activation chemistry for continuous and accurate VCG replication.
虚拟环状基因组 (VCG) 模型被提出作为超越模板复制的一种手段，以实现生命起源中非酶 RNA 复制的无限循环。在 VCG 模型中，原细胞基因组是一组短的寡核苷酸，映射到虚拟环状序列的两条链上。复制是由一组动力学捕获的部分碱基配对构象上的模板非酶引物延伸驱动的，然后这些构象被混合以实现寡核苷酸的持续延伸。在这里，我们描述了对 VCG 模型进行复制的可行性的初步实验研究。我们设计了一个小的 12 个核苷酸的模型 VCG，并合成了所有长度为 2 到 12 的 247 个寡核苷酸，对应于这个基因组。我们在添加激活核苷酸后，实验监测了标记引物在 VCG 寡核苷酸池中的个体命运，并研究了寡核苷酸长度、浓度、组成和温度等因素对引物延伸程度的影响。我们观察到 VCG 系统中一个惊人的延长平衡过程，使反应能够进行相当大的程度。我们发现，由于最短的寡核苷酸只能在低温下与模板结合，而最长的寡核苷酸需要高温尖峰才能从非活性构象中逃脱，因此环境波动对于整个 VCG 系统的连续模板延伸至关重要。最后，我们证明了当 VCG 寡核苷酸混合物预先激活时，引物延伸显著增强。我们讨论了持续和准确的 VCG 复制所需的原位激活化学的必要性。
