title: Linear molecular beacons for highly sensitive bioanalysis based on cyclic Exo III enzymatic amplification.
abstract: Sensitive analysis or monitoring of biomolecules and small molecules is very important for many biological researches, clinical diagnosis and forensic investigations. As a sequence-independent exonuclease, Exonuclease III (Exo III) has been widely used for amplified detection of proteins and nucleic acids where displacing probes or molecular beacons are used as the signaling probes. However, displacing probes suffer slow hybridization rate and high background signal and molecular beacons are difficult to design and prone to undesired nonspecific interactions. Herein, we report a new type of probes called linear molecular beacons (LMBs) for use in Exo III amplification assays to improve hybridization kinetics and reduce background noises. LMBs are linear oligonucleotide probes with a fluorophore and quencher attached to 3' terminal and penultimate nucleotides, respectively. Compared to conventional molecular beacons and displacing probes, LMBs are easy to design and synthesize. More importantly, LMBs have a much lower background noise and allow faster reaction rates. Using LMBs in cyclic Exo III amplification assay, ultrasensitive nucleic acid detection methods were developed with a detection limit of less than 120fM, which is 2 orders of magnitude lower than that of conventional molecular beacons or displacing probes-based Exo III amplification assays. Furthermore, LMBs can be extended as universal probes for detection of non-nucleic acid molecules such as cocaine with high sensitivity. These results demonstrate that the combination of Exo III amplification and LMB signaling provides a general method for ultrasensitive and selective detection of a wide range of targets.
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, PR China. cyyang@xmu.edu.cn
authors.forename: Chaoyong James
authors.initials: CJ
authors.lastname: Yang
authors.forename: Liang
authors.initials: L
authors.lastname: Cui
authors.forename: Jiahao
authors.initials: J
authors.lastname: Huang
authors.forename: Ling
authors.initials: L
authors.lastname: Yan
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Lin
authors.forename: Chunming
authors.initials: C
authors.lastname: Wang
authors.forename: Wei Yun
authors.initials: WY
authors.lastname: Zhang
authors.forename: Huaizhi
authors.initials: H
authors.lastname: Kang
mesh_terms: D003042:Cocaine / Q000032:analysis*; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D009696:Nucleic Acids / Q000032:analysis*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D003042:Cocaine