Saxena Sarika, Joshi Savita, Shankaraswamy J, Tyagi Shikhar, Kukreti Shrikant
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, 201313, India.
Department of Chemistry, Nucleic Acids Research laboratory, University of Delhi (North Campus), Delhi, 110007, India.
Biopolymers. 2017 Jul;107(7). doi: 10.1002/bip.23018.
Most of the important genomic regions, especially the G,C rich gene promoters, consist of sequences with potential to form G,C-tetraplexes on both the DNA strands. In this study, we used three C-rich oligonucleotides (11Py, 21Py, and HTPy), of which 11Py and 21Py are located at various transcriptional regulatory elements of the human genome while HTPy sequence is a C-rich strand of human telomere sequence. These C-rich oligonucleotides formed i-motif structures, verified by Circular Dichroism (CD), UV absorption melting experiments, and native gel electrophoresis. The CD spectra revealed that 11Py and 21Py form i-motif structures at acidic pH values of 4.5 and 5.7 in the presence of 100 mM NaCl but remain unstructured at pH 7.0. However, 21Py can form stable i-motif structure even at neutral pH in presence of 1 mM MgCl . UV-thermal melting studies showed stabilization of 21Py i-motif at pH 5.7 in the presence of Na or K with increasing concentration of MgCl or CaCl from 1 to 10 mM. Significant shift in the CD peak of HTPy sequence was observed as the positive peak from 286 nm shifted to 276 nm while the negative peak from 265 to 254 nm. Further, inevitable necessity of 1 mM Mg to form i-motif structure at neutral pH was observed. Under similar ionic conditions and neutral pH, all the three C-rich sequences were able to form stable i-motif structures (11Py, 21Py) or altered i-motif/homoduplex structures (HTPy) in the presence of MgCl and cell mimicking molecular crowding conditions of 40 wt% PEG 200. It is concluded that presence of Mg ions and molecular crowding agents induce and stabilize i-motif structures at physiological solution environment.
大多数重要的基因组区域，尤其是富含G、C的基因启动子，由在两条DNA链上都有可能形成G、C-四链体的序列组成。在本研究中，我们使用了三种富含C的寡核苷酸（11Py、21Py和HTPy），其中11Py和21Py位于人类基因组的各种转录调控元件上，而HTPy序列是人类端粒序列的富含C的链。这些富含C的寡核苷酸形成了i-基序结构，通过圆二色性（CD）、紫外吸收熔解实验和非变性凝胶电泳进行了验证。CD光谱显示，在100 mM NaCl存在的情况下，11Py和21Py在酸性pH值4.5和5.7时形成i-基序结构，但在pH 7.0时保持无结构状态。然而，即使在中性pH值且存在1 mM MgCl₂的情况下，21Py也能形成稳定的i-基序结构。紫外热熔解研究表明，在pH 5.7时，随着MgCl₂或CaCl₂浓度从1 mM增加到10 mM，Na⁺或K⁺的存在会使21Py的i-基序结构稳定。观察到HTPy序列的CD峰有明显位移，正峰从286 nm移至276 nm，负峰从265 nm移至254 nm。此外，还观察到在中性pH值下形成i-基序结构不可避免地需要1 mM Mg²⁺。在相似的离子条件和中性pH值下，在MgCl₂存在以及40 wt% PEG 200模拟细胞的分子拥挤条件下，所有这三种富含C的序列都能够形成稳定的i-基序结构（11Py、21Py）或改变的i-基序/同源双链结构（HTPy）。得出的结论是，Mg²⁺离子和分子拥挤剂在生理溶液环境中诱导并稳定i-基序结构。
