A label-free, quadruplex-based functional molecular beacon (LFG4-MB) for fluorescence turn-on detection of DNA and nuclease.

We demonstrate a novel concept for the construction of a label-free, quadruplex-based functional molecular beacon (LFG4-MB) by using G-quadruplex motif as a substitute for Watson-Crick base pairing in the MB stem and a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM) as a reporter. It shows high sensitivity in assays for UDG activity/inhibition and detection of DNA sequence based on the unique fluorescence increase that occurs as a result of the strong interaction between NMM and the folded quadruplex upon removal of uracil by UDG or displacement of block sequence by target DNA. The LFG4-MB is simple in design, fast in operation and could be easily transposed to other biological relevant target analysis by simply changing the recognition portion. The LFG4-MB does not require any chemical modification for DNA, which offers the advantages of simplicity and cost efficiency and obviates the possible interference with the affinity and specificity of the MB as well as the kinetic behavior of the catalysts caused by the bulky fluorescent groups. More importantly, the LFG4-MB offers great extent of freedom to tune the experimental conditions for the general applicability in bioanalysis.

[1]  B. Liu,et al.  Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes. , 2007, Analytical biochemistry.

[2]  H. Nilsen,et al.  Base excision repair in a network of defence and tolerance. , 2001, Carcinogenesis.

[3]  P. Bolton,et al.  Fluorescent dyes specific for quadruplex DNA. , 1998, Nucleic acids research.

[4]  J. Mergny,et al.  Quadruplex-based molecular beacons as tunable DNA probes. , 2006, Journal of the American Chemical Society.

[5]  Jean-Louis Mergny,et al.  DNA duplex–quadruplex exchange as the basis for a nanomolecular machine , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[7]  Hao Wang,et al.  Strategy for molecular beacon binding readout: separating molecular recognition element and signal reporter. , 2009, Analytical chemistry.

[8]  Xiaogang Qu,et al.  Design of proton-fueled tweezers for controlled, multi-function DNA-based molecular device. , 2010, Biochimie.

[9]  Huang-Hao Yang,et al.  Increasing the sensitivity and single-base mismatch selectivity of the molecular beacon using graphene oxide as the "nanoquencher". , 2010, Chemistry.

[10]  Fred Russell Kramer,et al.  Multicolor molecular beacons for allele discrimination , 1998, Nature Biotechnology.

[11]  Stephen Neidle,et al.  Crystal structure of parallel quadruplexes from human telomeric DNA , 2002, Nature.

[12]  J. Tainer,et al.  A nucleotide-flipping mechanism from the structure of human uracil–DNA glycosylase bound to DNA , 1996, Nature.

[13]  Friedrich C Simmel,et al.  A DNA-based machine that can cyclically bind and release thrombin. , 2004, Angewandte Chemie.

[14]  J. Chaires,et al.  Sequence and structural selectivity of nucleic acid binding ligands. , 1999, Biochemistry.

[15]  Itamar Willner,et al.  DNAzymes for sensing, nanobiotechnology and logic gate applications. , 2008, Chemical Society reviews.

[16]  J. Chaires,et al.  Tiny telomere DNA. , 2002, Nucleic acids research.

[17]  A. Maksimenko,et al.  A molecular beacon assay for measuring base excision repair activities. , 2004, Biochemical and biophysical research communications.

[18]  Xiaogang Qu,et al.  Sensitive, selective and label-free protein detection using a smart polymeric transducer and aptamer/ligand system. , 2009, Chemical communications.

[19]  Kemin Wang,et al.  Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction , 2009, Nucleic acids research.

[20]  E. Carpenter,et al.  A Comparative Study of Uracil-DNA Glycosylases from Human and Herpes Simplex Virus Type 1* , 2005, Journal of Biological Chemistry.

[21]  R. Osman,et al.  Role of DNA flexibility in sequence-dependent activity of uracil DNA glycosylase. , 2002, Biochemistry.

[22]  N. Sugimoto,et al.  Artificial G-wire switch with 2,2'-bipyridine units responsive to divalent metal ions. , 2007, Journal of the American Chemical Society.

[23]  J. Rouillard,et al.  Sensitive and Selective Label-Free DNA Detection by Conjugated Polymer-Based Microarrays and Intercalating Dye , 2008 .

[24]  X. Xie,et al.  Enzymatic signal amplification of molecular beacons for sensitive DNA detection , 2008, Nucleic acids research.

[25]  Andreas Reuter,et al.  Eine DNA‐basierte Maschine, die Thrombin abwechselnd binden und wieder freigeben kann , 2004 .

[26]  R. Fahlman,et al.  Cation-regulated self-association of "synapsable" DNA duplexes. , 1998, Journal of molecular biology.

[27]  Samuel H. Wilson,et al.  Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase β and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity , 2005, Nucleic acids research.

[28]  Xiaogang Qu,et al.  A DNA nanomachine induced by single-walled carbon nanotubes on gold surface. , 2009, Biomaterials.

[29]  N. Broude,et al.  Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology. , 2002, Trends in biotechnology.

[30]  DNA-small molecule chimera with responsive protein-binding ability. , 2008, Journal of the American Chemical Society.

[31]  S. Neidle,et al.  Structure of the first parallel DNA quadruplex-drug complex. , 2003, Journal of the American Chemical Society.

[32]  A. Libchaber,et al.  Single-mismatch detection using gold-quenched fluorescent oligonucleotides , 2001, Nature Biotechnology.

[33]  J. Stivers,et al.  2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects. , 1998, Nucleic acids research.

[34]  K. Plaxco,et al.  Sensitive and selective amplified fluorescence DNA detection based on exonuclease III-aided target recycling. , 2010, Journal of the American Chemical Society.

[35]  L. Gros,et al.  Enzymology of the repair of free radicals-induced DNA damage , 2002, Oncogene.

[36]  Itamar Willner,et al.  Electronic aptamer-based sensors. , 2007, Angewandte Chemie.

[37]  X. Qu,et al.  A quadruplex-based, label-free, and real-time fluorescence assay for RNase H activity and inhibition. , 2010, Chemistry.

[38]  Xiaogang Qu,et al.  Artificial DNA Nano‐Spring Powered by Protons , 2010, Advanced materials.

[39]  A. Lane,et al.  Stability and kinetics of G-quadruplex structures , 2008, Nucleic acids research.

[40]  Ronghua Yang,et al.  Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization. , 2008, Journal of the American Chemical Society.

[41]  Yi Xiao,et al.  i-Motif quadruplex DNA-based biosensor for distinguishing single- and multiwalled carbon nanotubes. , 2009, Journal of the American Chemical Society.