Modulating molecular level space proximity: a simple and efficient strategy to design structured DNA probes.
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/gamma-CD induced by 1.0 equiv target DNA is near 174, which is 4-fold higher than that in the absence of gamma-CD. In addition, the usage of gamma-CD shifts the melting temperature of P1 from 57 to 68 degrees 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.
Zheng
Jing
J
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China.
Li
Jishan
J
Gao
Xiaoxia
X
Jin
Jianyu
J
Wang
Kemin
K
Tan
Weihong
W
Yang
Ronghua
R
D004396:Coloring Agents / Q000737:chemistry*; D003505:Cyclodextrins / Q000737:chemistry; D015342:DNA Probes / Q000138:chemical synthesis* / Q000737:chemistry; D008968:Molecular Conformation
D004396:Coloring Agents; D003505:Cyclodextrins; D015342:DNA Probes