Martí Angel A, Jockusch Steffen, Stevens Nathan, Ju Jingyue, Turro Nicholas J
Department of Chemistry, Columbia University, New York, NY 10027, USA.
Acc Chem Res. 2007 Jun;40(6):402-9. doi: 10.1021/ar600013q. Epub 2007 Apr 25.
We outline the different approaches taken by our group in the design of fluorescent hybridization sensors. Molecular beacons (MBs) and binary probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.
我们概述了本研究团队在设计荧光杂交传感器时所采用的不同方法。已合成了分别使用两种染料的分子信标（MBs）和二元探针（BPs）（分别为2d-MB和2d-BP）；这些传感器在与靶标生物分子（如DNA）结合时可作为发射开关。这些传感器通过可视化与靶标多核苷酸（PN）结合时的探针，实现对多核苷酸的比率荧光检测。此外，还开发了三染料MBs（3d-MB）和BPs（3d-BP），其中采用能量转移级联来减少荧光团发射光谱之间的重叠，从而降低受体荧光团的直接激发。还合成了具有长荧光寿命优势的芘基MB（Py-MB）和BP（Py-BP）。时间分辨荧光光谱（TRES）可用于区分芘探针的短寿命背景荧光和长寿命荧光。通过对加州海兔细胞提取物中Py-BP的信号与背景荧光进行时间分辨，证明了该技术。
