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Modulating Molecular Level Space Proximity: A Simple and Efficient Strategy to Design Structured DNA Probes

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journal contribution
posted on 01.05.2010, 00:00 authored by Jing Zheng, Jishan Li, Xiaoxia Gao, Jianyu Jin, Kemin Wang, Weihong Tan, Ronghua Yang
To construct efficient oligonucleotide probes, specific nucleic acid is designed as a conformationally constrained form based on the formation of a Watson−Crick-based duplex. However, instability of Watson−Crick hydrogen bonds in a complex biological environment usually leads to high background signal from the probe itself and false positive signal caused by nonspecific binding. To solve this problem, we propose a way to restrict the labeled-dyes in a hydrophobic cavity of cyclodextrin. This bounding, which acts like extra base pairs to form the Watson−Crick duplex, achieves variation of level of space proximity of the two labels and thus the degree of conformational constraint. To demonstrate the feasibility of the design, a stem-containing oligonucleotide probe (P1) for DNA hybridization assay and a stemless one (P2) for protein detection were examined as models. Both oligonucleotides were doubly labeled with pyrene at the 5′- and 3′- ends, respectively. It is the cyclodextrin/pyrene inclusion interaction that allows modulating the degree of conformational constraints of P1 and P2 and thus their background signals and selectivity. Under the optimal conditions, the ratio of signal-to-background of P1/γ-CD induced by 1.0 equiv target DNA is near 174, which is 4-fold higher than that in the absence of γ-CD. In addition, the usage of γ-CD shifts the melting temperature of P1 from 57 to 68 °C, which is reasonable for improving target-binding selectivity. This approach is simple in design, avoiding any variation of the stem’s length and sequences. Furthermore, the strategy is generalizable which is suited for not only the stem-containing probe but also the linear probe with comparable sensitivity and selectivity to conventional structured DNA probes.

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