Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, California 91125, United States.
J Am Chem Soc. 2017 Dec 6;139(48):17301-17304. doi: 10.1021/jacs.7b10639. Epub 2017 Nov 21.
We report a bifunctional fluorescent probe that combines a rhodium metalloinsertor with a cyanine dye as the fluorescent reporter. The conjugate shows weak luminescence when free in solution or with well matched DNA but exhibits a significant luminescence increase in the presence of a 27-mer DNA duplex containing a central CC mismatch. DNA photocleavage experiments demonstrate that, upon photoactivation, the conjugate cleaves the DNA backbone specifically near the mismatch site on a 27-mer fragment, consistent with mismatch targeting. Fluorescence titrations with the 27-mer duplex containing the CC mismatch reveal a DNA binding affinity of 3.1 × 10 M, similar to that of other rhodium metalloinsertors. Fluorescence titrations using genomic DNA extracted from various cell lines demonstrate a clear discrimination in fluorescence between those cell lines that are proficient or deficient in mismatch repair. This differential luminescence reflects the sensitive detection of the mismatchrepair-deficient phenotype.
我们报告了一种双功能荧光探针，它将铑金属插入剂与菁染料作为荧光报告基团结合在一起。当游离在溶液中或与匹配良好的 DNA 结合时，该缀合物的发光很弱，但在存在含有中央 CC 错配的 27 个碱基对 DNA 双链体时，发光强度显著增加。DNA 光解实验表明，在光激活后，该缀合物在 27 个碱基对片段上的错配位点附近特异性地切割 DNA 骨架，与错配靶向一致。用含有 CC 错配的 27 个碱基对双链体进行荧光滴定，揭示出 DNA 结合亲和力为 3.1×10^M，与其他铑金属插入剂相似。使用从各种细胞系提取的基因组 DNA 进行荧光滴定，清楚地区分了那些在错配修复中有效或无效的细胞系。这种差异荧光反映了对错配修复缺陷表型的灵敏检测。
