Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States.
J Am Chem Soc. 2020 Oct 14;142(41):17766-17781. doi: 10.1021/jacs.0c08996. Epub 2020 Oct 5.
Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'--methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.
控制核酸的结构和活性极大地扩展了它们在治疗、生物传感、纳米技术和生物计算中的应用潜力。已经开发了几种方法来赋予 DNA 和 RNA 对小分子和基于光的刺激的响应性。然而，热触发的核酸控制在很大程度上仍未得到探索，这使得响应性核酸技术存在显著差距。此外，当前的技术仅限于天然核酸，并且通常与聚合酶产生的序列不兼容。在这里，我们表明乙二醛是一种经过充分研究的化合物，它可以共价连接到几个碱基的沃森-克里克-富兰克林面上，通过热可逆地调节几乎任何核酸支架的结构和活性来解决这些限制。使用各种 DNA 和 RNA 构建体，我们证明了乙二醛修饰很容易进行，并能强烈破坏核酸的结构和功能。我们还对脱笼动力学进行了表征，并表明在各种条件下，可以通过可调节的热去除乙二醛加合物来恢复活性。我们还通过可逆地笼封 2'--甲基化 RNA 适体以及合成的 threose nucleic acid (TNA) 和 peptide nucleic acid (PNA) 支架进一步说明了这种方法的多功能性。乙二醛笼封也可用于可逆地破坏酶-核酸相互作用，我们表明向导 RNA 的笼封允许对 CRISPR-Cas9 活性进行可调且可逆的控制。我们还证明了乙二醛笼封作为一种有效方法来提高 PCR 特异性，并且我们笼封了一种生物稳定的反义寡核苷酸，以在活细胞中进行基因表达的时间释放激活和滴定。总之，乙二醛化是一种简单而无痕的方法，可以赋予理论上任何核酸结构的可逆热响应性，这在合成生物学中是一个重要的需求，并为在医学、纳米技术和生物计算中构建可编程核酸组件提供了一种通用的新工具。
