Zhang Lihua, Liu Hua, Shao Yong, Lin Clement, Jia Huan, Chen Gang, Yang Danzhou, Wang Ying
Institute of Physical Chemistry, Zhejiang Normal University , Jinhua, Zhejiang 321004, China.
Anal Chem. 2015 Jan 6;87(1):730-7. doi: 10.1021/ac503730j. Epub 2014 Dec 9.
Aptamers, that exist naturally in living cells as functional elements and can switch nonfluorescent natural targets to fluorophores, are very useful in developing highly sensitive and selective biosensors and screening functional agents. This work demonstrates that human telomeric G-quadruplex (HTG) can serve as a potential fluorophore-switching aptamer (FSA) to target a natural isoquinoline alkaloid. We found that, among the G-quadruplexes studied here and the various structurally similar alkaloids including epiberberine (EPI), berberine (BER), palmatine (PAL), jatrorrhizine (JAT), coptisine (COP), worenine (WOR), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT), only the HTG DNA, especially with a 5'-TA-3' residue at the 5' end of the G-quadruplex tetrad (5'-TAG3(TTAG3)3-3', TA[Q]) as the minimal sequence, is the most efficient FSA to selectively light up the EPI fluorescence. Compared to the 5' end flanking sequences, the 3' end flanking sequences of the tetrad contribute significantly less to the recognition of EPI. The binding affinity of EPI to TA[Q] (K(d) = 37 nM) is at least 20 times tighter than those of the other alkaloids. The steady-state absorption, steady-state/time-resolved fluorescence, and NMR studies demonstrate that EPI most likely interact with the 5' end flanking sequence substructure beyond the core [Q] and the G-quadruplex tetrad in a much more specific manner than the other alkaloids. The highly selective and tight binding of EPI with the FSA and significantly enhanced fluorescence suggest the potential development of a selective EPI sensor (detection limit of 10 nM). More importantly, EPI, as the brightest FSA emitter among the alkaloids, can also serve as an efficient conformation probe for HTG DNA and discriminate the DNA G-quadruplex from the RNA counterpart. Furthermore, EPI can bind stoichiometrically to each G-quadruplex unit of long HTG DNA multimer with the most significant fluorescence enhancement, which has not been achieved by the previously reported probes. Our work suggests the potential use of EPI as a bioimaging probe and a therapeutic DNA binder.
适体作为活细胞中天然存在的功能元件，能够将无荧光的天然靶标转变为荧光团，在开发高灵敏度和高选择性生物传感器以及筛选功能试剂方面非常有用。这项工作表明，人端粒G-四链体（HTG）可作为一种潜在的荧光团转换适体（FSA）来靶向一种天然异喹啉生物碱。我们发现，在此研究的G-四链体以及各种结构相似的生物碱中，包括表小檗碱（EPI）、小檗碱（BER）、巴马汀（PAL）、药根碱（JAT）、黄连碱（COP）、吴茱萸碱（WOR）、血根碱（SAN）、白屈菜红碱（CHE）和两面针碱（NIT），只有HTG DNA，特别是在G-四链体四分体5'端带有5'-TA-3'残基（5'-TAG3(TTAG3)3-3'，TA[Q]）作为最小序列时，是最有效的FSA，能够选择性地激发EPI的荧光。与5'端侧翼序列相比，四分体的3'端侧翼序列对EPI的识别贡献显著较小。EPI与TA[Q]的结合亲和力（K(d)=37 nM）比其他生物碱至少高20倍。稳态吸收、稳态/时间分辨荧光和核磁共振研究表明，EPI与核心[Q]和G-四链体四分体之外的5'端侧翼序列亚结构的相互作用方式比其他生物碱更具特异性。EPI与FSA的高度选择性和紧密结合以及显著增强的荧光表明，有可能开发出一种选择性EPI传感器（检测限为10 nM）。更重要的是，EPI作为生物碱中最亮的FSA发射体，还可作为HTG DNA的有效构象探针，区分DNA G-四链体和RNA对应物。此外，EPI可以化学计量地与长HTG DNA多聚体的每个G-四链体单元结合，荧光增强最为显著，这是以前报道的探针所未实现的。我们的工作表明EPI有作为生物成像探针和治疗性DNA结合剂的潜在用途。
