G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.
G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.
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DOI：
10.1093/nar/gkv749
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发表时间：
2015-09-18
影响因子：
14.9
通讯作者：
Myong S
中科院分区：
文献类型：
--
作者：
Kreig A;Calvert J;Sanoica J;Cullum E;Tipanna R;Myong S
G-四链体（GQ）是替代的DNA二级结构，可以在整个人类基因组中形成并控制重要调节基因的复制和转录。在这里，我们建立了一个合奏的荧光分析，采用两个GQ相互作用的化合物，N-甲基中卟啉IX（NMM）和结晶紫（CV）。这使得能够定量测量单链（ss）和双链（ds）DNA中的GQ折叠倾向和构象特异性。我们的GQ作图表明，GQ形成的可能性在dsDNA中大大降低，这可能是由于沃森-克里克碱基配对的竞争。与ssDNA中形成平行和反平行GQ的GQ折叠序列不同，dsDNA仅显示平行折叠。此外，我们采用单分子FRET获得稳定形成的，弱折叠和未折叠的GQ构象的直接定量。这项研究的结果和这里开发的方法将能够识别和分类人类基因组中潜在的GQ形成序列。
G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.