Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging
Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging
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DOI：
10.1021/jacs.0c08996
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发表时间：
2020-10-14
影响因子：
15
通讯作者：
Heemstra, Jennifer M.
中科院分区：
文献类型：
--
作者：
Knutson, Steve D.;Sanford, Aimee A.;Heemstra, Jennifer M.
控制核酸的结构和活性极大地扩展了它们在治疗学、生物传感、纳米技术和生物计算方面的应用潜力。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反应性。然而，核酸的热触发控制在很大程度上仍未被探索，在响应式核酸技术上留下了重大空白。此外，目前的技术仅限于天然核酸，往往与聚合酶产生的序列不相容。在这里，我们展示了乙二醛，一种很好的表征化合物，共价地附着在几个核碱基的沃森-克里克-富兰克林面，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。使用多种DNA和RNA结构，我们证明乙二醛修饰很容易安装，并有效地破坏核酸的结构和功能。我们还描述了衰减的动力学，并表明在各种条件下，通过可调的热去除乙二醛加合物可以恢复活性。我们通过可逆笼化2'- o甲基化RNA适配体以及合成的蔗糖核酸（TNA）和肽核酸（PNA）支架进一步说明了这种方法的多功能性。乙二醛笼化也可用于可逆地破坏酶-核酸相互作用，我们表明，引导RNA的笼化允许对CRISPR-Cas9活性进行可调和可逆的控制。我们还证明了乙二醛笼是提高PCR特异性的有效方法，我们笼了一个生物稳定的反义寡核苷酸，用于活细胞中基因表达的时间释放激活和滴定。总之，乙草醛化是一种直接的、无星形的方法，可以对理论上任何核酸结构赋予可逆的热响应性，解决了合成生物学中的重要需求，并为构建医学、纳米技术和生物计算中的可编程核酸组分提供了一种多功能的新工具。
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'-O-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 starless 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.