Hnedzko Dziyana, McGee Dennis W, Karamitas Yannis A, Rozners Eriks
Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York 13902, USA.
Department of Biological Sciences, Binghamton University, State University of New York, Binghamton, New York 13902, USA.
RNA. 2017 Jan;23(1):58-69. doi: 10.1261/rna.058362.116. Epub 2016 Oct 14.
Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research, and biotechnology. However, specific recognition of structured RNA is challenging, and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well-suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (K = 9.4 to 17 × 10 M) and sequence-selective triple helices with RNA hairpins at physiological pH and salt concentration. The affinity of PNA-peptide conjugates for the matched RNA hairpins was unusually high compared to the much lower affinity for DNA hairpins of the same sequence (K = 0.05 to 1.1 × 10 M). The binding of double-stranded RNA by M-modified PNA-peptide conjugates was a relatively fast process (k = 2.9 × 10 M sec) compared to the notoriously slow triple helix formation by oligodeoxynucleotides (k ∼ 10 M sec). M-modified PNA-peptide conjugates were not cytotoxic and were efficiently delivered in the cytosol of HEK293 cells at 10 µM. Surprisingly, M-modified PNAs without peptide conjugation were also taken up by HEK293 cells, which, to the best of our knowledge, is the first example of heterocyclic base modification that enhances the cellular uptake of PNA. Our results suggest that M-modified PNA-peptide conjugates are promising probes for sequence-selective recognition of double-stranded RNA in live cells and other biological systems.
对活细胞中复杂RNA进行序列选择性识别在生物学、生物医学研究和生物技术领域有着广泛的应用前景。然而，对结构化RNA进行特异性识别具有挑战性，且缺乏普遍适用且有效的方法。最近，我们发现肽核酸（PNA）是识别双链RNA异常合适的配体。在此，我们报告2-氨基吡啶（M）修饰的PNA及其与赖氨酸和精氨酸三肽的缀合物在生理pH和盐浓度下与RNA发夹形成强（K = 9.4至17×10 M）且序列选择性的三链螺旋。与对相同序列的DNA发夹的低得多的亲和力（K = 0.05至1.1×10 M）相比，PNA-肽缀合物对匹配的RNA发夹的亲和力异常高。与寡脱氧核苷酸形成三链螺旋的极其缓慢的过程（k ∼ 10 M sec）相比，M修饰的PNA-肽缀合物与双链RNA的结合是一个相对快速的过程（k = 2.9×10 M sec）。M修饰的PNA-肽缀合物没有细胞毒性，并且在10 µM时能有效地递送至HEK293细胞的胞质溶胶中。令人惊讶的是，未与肽缀合的M修饰的PNA也能被HEK293细胞摄取，据我们所知，这是杂环碱基修饰增强PNA细胞摄取的首个例子。我们的结果表明，M修饰的PNA-肽缀合物是用于在活细胞和其他生物系统中对双链RNA进行序列选择性识别的有前途的探针。
