Sabale Pramod M, Ambi Uddhav B, Srivatsan Seergazhi G
Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune, Doctor Homi Bhabha Road, Pune 411008, India.
ACS Omega. 2018 Nov 30;3(11):15343-15352. doi: 10.1021/acsomega.8b02550. Epub 2018 Nov 12.
The ability to bind strongly to complementary nucleic acid sequences, invade complex nucleic acid structures, and resist degradation by cellular enzymes has made peptide nucleic acid (PNA) oligomers as very useful hybridization probes in molecular diagnosis. For such applications, the PNA oligomers have to be labeled with appropriate reporters as they lack intrinsic labels that can be used in biophysical assays. Although solid-phase synthesis is commonly used to attach reporters onto PNA, development of milder and modular labeling methods will provide access to PNA oligomers labeled with a wider range of biophysical tags. Here, we describe the establishment of a postsynthetic modification strategy based on bioorthogonal chemical reactions in functionalizing PNA oligomers in solution with a variety of tags. A toolbox composed of alkyne- and azide-modified monomers were site-specifically incorporated into PNA oligomers and postsynthetically click-functionalized with various tags, ranging from sugar, amino acid, biotin, to fluorophores, by using copper(I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, and Staudinger ligation reactions. As a proof of utility of this method, fluorescent PNA hybridization probes were developed and used in imaging human telomeres in chromosomes and poly(A) RNAs in cells. Taken together, this simple approach of generating a wide range of functional PNA oligomers will expand the use of PNA in molecular diagnosis.
肽核酸（PNA）寡聚物能够与互补核酸序列紧密结合，侵入复杂的核酸结构，并抵抗细胞酶的降解，这使其成为分子诊断中非常有用的杂交探针。对于此类应用，PNA寡聚物必须用合适的报告基团进行标记，因为它们缺乏可用于生物物理分析的固有标记。尽管固相合成通常用于将报告基团连接到PNA上，但开发更温和、模块化的标记方法将能够获得用更广泛的生物物理标签标记的PNA寡聚物。在此，我们描述了一种基于生物正交化学反应的合成后修饰策略，用于在溶液中用各种标签对PNA寡聚物进行功能化。一个由炔烃和叠氮化物修饰的单体组成的工具箱被位点特异性地掺入PNA寡聚物中，并通过使用铜（I）催化的叠氮化物-炔烃环加成、应变促进的叠氮化物-炔烃环加成和施陶丁格连接反应，与从糖、氨基酸、生物素到荧光团等各种标签进行合成后点击功能化。作为该方法实用性的证明，开发了荧光PNA杂交探针，并用于对染色体中的人类端粒和细胞中的聚（A）RNA进行成像。综上所述，这种生成多种功能性PNA寡聚物的简单方法将扩大PNA在分子诊断中的应用。
