Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes.
Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes.
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DOI：
10.1002/chem.202103616
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发表时间：
2022-01-10
期刊：
影响因子：
--
通讯作者：
Bong D
中科院分区：
文献类型：
--
作者：
Liang Y;Miao S;Mao J;Devari S;Gonzalez M;Bong D
我们假设与非规范核酸基序的可编程杂交可以通过大分子展示单个非规范对（NCP）的结合物来实现。由于每个识别元件可能单独与 NCP 具有较弱的结合，因此我们开发了一种半理性方法来检测双链 DNA 和 RNA 中选定的含氮碱基与非规范位点之间的低亲和力相互作用。通过将非生物（三嗪、嘧啶）和天然 RNA 碱基与噻唑橙 (TO) 染料偶联来合成一组荧光探针。该探针文库针对带有单个脱碱基、单个 NCP 和串联 NCP 位点的双链体核酸底物进行筛选。据报道，探针与 NCP 位点的结合比背景荧光增强了 100-1000 倍。结合强烈依赖于上下文，反映了分子识别和稳定性：不太稳定的基序更有可能结合合成探针。此外，DNA 和 RNA 底物表现出完全不同的脱碱基和单一 NCP 结合谱。虽然无碱基和单一 NCP 筛选中的探针结合是单调的，但通过 RNA 中串联 NCP 位点的筛选观察到更丰富的结合谱，部分原因是空间可及性增加。除了三嗪三聚氰胺 (M) 和 T/U 位点之间已知的结合相互作用之外，NCP 筛选还确定了单个 NCP 和 2×2 内部凸起中富含嘧啶基序的新靶向元件。我们预计这种类型的半理性方法将导致大分子水平上的可编程非规范杂交策略。我们开发了一种半理性方法来检测双链 DNA 和 RNA 中与非规范对 (NCP) 位点的低亲和力结合。针对具有单脱碱基、单 NCP 和串联 NCP 位点的 dsDNA 和 dsRNA 筛选一组带有非生物（三嗪、嘧啶）和天然 RNA 碱基的荧光噻唑橙探针。荧光探针的结合揭示了与 NCP 的新颖且选择性的碱基相互作用，为靶向 RNA 中非规范基序的新策略奠定了基础。
We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000X fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level. We have developed a semi-rational approach to detect low affinity binding to noncanonical pair (NCP) sites in duplex DNA and RNA. A set of fluorogenic thiazole orange probes bearing abiotic (triazines, pyrimidines) and native RNA bases was screened against dsDNAs and dsRNAs with single abasic, single NCP and tandem NCP sites. Fluorogenic probe engagement revealed novel and selective base interactions with NCPs, setting the stage for a new strategy of targeting noncanonical motifs in RNA.
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