Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids.
Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids.
复制标题
DOI：
10.1261/rna.058362.116
复制
发表时间：
2017-01
期刊：
影响因子：
--
通讯作者：
Rozners E
中科院分区：
文献类型：
--
作者：
Hnedzko D;McGee DW;Karamitas YA;Rozners E
活细胞中复杂RNA的序列选择性识别可以在生物学、生物医学研究和生物技术中找到广泛的应用。然而，结构化RNA的特异性识别是具有挑战性的，并且缺乏普遍适用且有效的方法。最近，我们发现肽核酸（PNAs）是非常适合识别双链RNA的配体。在此，我们报告了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（Ka = 9.4至17 × 107 M−1）和序列选择性的三螺旋。PNA-肽缀合物对匹配的RNA发夹的亲和力异常高，而对相同序列的DNA发夹的亲和力要低得多（Ka = 0.05至1.1 × 107 M−1）。与寡脱氧核苷酸形成三股螺旋的速度（kon = 103 M− 1 sec−1）相比，M修饰的PNA-肽缀合物与双链RNA的结合是一个相对较快的过程（kon = 2.9 × 104 M− 1 sec −1）。M-修饰的PNA-肽缀合物没有细胞毒性，并且在10 μM时有效地递送到HEK 293细胞的胞质溶胶中。令人惊讶的是，没有肽缀合的M-修饰的PNA也被HEK 293细胞摄取，据我们所知，这是增强PNA的细胞摄取的杂环碱基修饰的第一个实例。我们的研究结果表明，M-修饰的PNA-肽缀合物是有前途的探针，用于活细胞和其他生物系统中的双链RNA的序列选择性识别。
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 (Ka = 9.4 to 17 × 107 M−1) 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 (Ka = 0.05 to 1.1 × 107 M−1). The binding of double-stranded RNA by M-modified PNA–peptide conjugates was a relatively fast process (kon = 2.9 × 104 M−1 sec−1) compared to the notoriously slow triple helix formation by oligodeoxynucleotides (kon ∼ 103 M−1 sec−1). 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.
登录
查看更多内容
影响因子：
15
作者：
Li, Ming;Zengeya, Thomas;Rozners, Eriks
通讯作者：
Rozners, Eriks
影响因子：
14.9
作者：
Hansen ME;Bentin T;Nielsen PE
通讯作者：
Nielsen PE
影响因子：
4.5
作者：
Fabani, Martin M.;Gait, Michael J.
通讯作者：
Gait, Michael J.
影响因子：
14.9
作者：
ESCUDE, C;FRANCOIS, JC;HELENE, C
通讯作者：
HELENE, C
影响因子：
5.8
作者：
Bonnet, E;Wuyts, J;Van de Peer, Y
通讯作者：
Van de Peer, Y