Imaging Sciences Training Program, Clinical Center and National Institute of Biomedical Imaging and Bioengineering, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2011 Sep 1;39(16):7114-23. doi: 10.1093/nar/gkr259. Epub 2011 May 18.
Guanine-rich sequences are highly abundant in the human genome, especially in regulatory regions. Because guanine-rich sequences have the unique ability to form G-quadruplexes, these structures may play a role in the regulation of gene transcription. In previous studies, we demonstrated that formation of G-quadruplexes could be induced with peptide nucleic acids (PNAs). PNAs designed to bind the C-rich strand upstream of the human BCL2 gene promoted quadruplex formation in the complementary G-rich strand. However, the question whether G-quadruplex formation was essential for PNA invasion remained unanswered. In this study, we compared PNA invasion in the native and mutant, i.e. not forming G-quadruplex, BCL2 sequences and showed that G-quadruplex is required for effective PNA invasion into duplex DNA. This finding provides strong evidence for not only sequence-specific, but also quadruplex specific, gene targeting with PNA probes. In addition, we examined DNA-duplex invasion potential of PNAs of various charges. Using the gel shift assay, chemical probing and dimethyl sulfate (DMS) protection studies, we determined that uncharged zwitterionic PNA has the highest binding specificity while preserving efficient duplex invasion.
富含鸟嘌呤的序列在人类基因组中高度丰富，特别是在调控区域。由于富含鸟嘌呤的序列具有形成 G-四链体的独特能力，这些结构可能在基因转录的调控中发挥作用。在之前的研究中，我们证明了肽核酸（PNA）可以诱导 G-四链体的形成。设计用于结合人类 BCL2 基因上游 C 丰富链的 PNA 促进了互补 G 丰富链中四链体的形成。然而，G-四链体的形成是否对 PNA 入侵至关重要的问题仍未得到解答。在这项研究中，我们比较了天然和突变（即不形成 G-四链体）的 BCL2 序列中的 PNA 入侵，并表明 G-四链体是 PNA 有效入侵双链 DNA 所必需的。这一发现不仅为 PNA 探针的序列特异性，也为四链体特异性基因靶向提供了强有力的证据。此外，我们还研究了各种电荷的 PNA 对 DNA 双链入侵的潜力。通过凝胶迁移分析、化学探测和二甲磺酸（DMS）保护研究，我们确定不带电荷的两性离子 PNA 具有最高的结合特异性，同时保持高效的双链入侵。
