Detection of bacterial 16S rRNA using a molecular beacon-based X sensor.

We demonstrate how a long structurally constrained RNA can be analyzed in homogeneous solution at ambient temperatures with high specificity using a sophisticated biosensor. The sensor consists of a molecular beacon probe as a signal reporter and two DNA adaptor strands, which have fragments complementary to the reporter and to the analyzed RNA. One adaptor strand uses its long RNA-binding arm to unwind the RNA secondary structure. Second adaptor strand with a short RNA-binding arm hybridizes only to a completely complementary site, thus providing high recognition specificity. Overall the three-component sensor and the target RNA form a four-stranded DNA crossover (X) structure. Using this sensor, Escherichia coli16S rRNA was detected in real time with the detection limit of ~0.17 nM. The high specificity of the analysis was proven by differentiating Bacillus subtilis from E. coli 16S rRNA sequences. The sensor responds to the presence of the analyte within seconds.

[1]  D. Kolpashchikov,et al.  A Single Molecular Beacon Probe Is Sufficient for the Analysis of Multiple Nucleic Acid Sequences , 2010, Chembiochem : a European journal of chemical biology.

[2]  David P. Kreil,et al.  Microarray oligonucleotide probes. , 2006, Methods in enzymology.

[3]  A. Ohashi,et al.  Peptide nucleic acids (PNAs) antisense effect to bacterial growth and their application potentiality in biotechnology , 2010, Applied Microbiology and Biotechnology.

[4]  Douglas R Call,et al.  Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays. , 2004, Biosensors & bioelectronics.

[5]  D. Ye,et al.  Molecular beacons: an optimal multifunctional biological probe. , 2008, Biochemical and biophysical research communications.

[6]  W. Pansegrau,et al.  Adhesion determinants of the Streptococcus species , 2009, Microbial biotechnology.

[7]  H. Jo,et al.  Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons , 2008, Nucleic acids research.

[8]  R. Amann,et al.  Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled Oligonucleotide Probes , 1998, Applied and Environmental Microbiology.

[9]  M. Moreno,et al.  Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development , 2012, Analytical and Bioanalytical Chemistry.

[10]  D. Kolpashchikov,et al.  Real-time SNP analysis in secondary-structure-folded nucleic acids. , 2010, Angewandte Chemie.

[11]  T. Tønjum,et al.  Molecular Diagnostics in Tuberculosis Basis and Implications for Therapy , 2012 .

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

[13]  D. Kolpashchikov,et al.  Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids. , 2011, Chemistry.

[14]  Darrell P. Chandler,et al.  Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays , 2003, Applied and Environmental Microbiology.

[15]  C. Keevil,et al.  Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms , 2006, Applied and Environmental Microbiology.

[16]  Darrell Chandler,et al.  Saliva‐Based Diagnostics Using 16S rRNA Microarrays and Microfluidics , 2007, Annals of the New York Academy of Sciences.

[17]  Sanjay Tyagi,et al.  Imaging intracellular RNA distribution and dynamics in living cells , 2009, Nature Methods.

[18]  Jingyue Ju,et al.  Fluorescent hybridization probes for nucleic acid detection , 2012, Analytical and Bioanalytical Chemistry.

[19]  K. Watanabe,et al.  Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation. , 1998, RNA.

[20]  Adam P. Silverman,et al.  Oligonucleotide probes for RNA-targeted fluorescence in situ hybridization. , 2007, Advances in clinical chemistry.

[21]  Shana O Kelley,et al.  Direct genetic analysis of ten cancer cells: tuning sensor structure and molecular probe design for efficient mRNA capture. , 2011, Angewandte Chemie.

[22]  Yulia V Gerasimova,et al.  DNA Nanotechnology for Nucleic Acid Analysis: DX Motif‐Based Sensor , 2011, Chembiochem : a European journal of chemical biology.

[23]  P. Nielsen,et al.  Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads , 2011, Artificial DNA, PNA & XNA.

[24]  G. Bao,et al.  Nanostructured Probes for RNA Detection in Living Cells , 2006, Annals of Biomedical Engineering.

[25]  Dmitry M Kolpashchikov,et al.  A binary DNA probe for highly specific nucleic Acid recognition. , 2006, Journal of the American Chemical Society.

[26]  D. Noguera,et al.  Making All Parts of the 16S rRNA of Escherichia coli Accessible In Situ to Single DNA Oligonucleotides , 2006, Applied and Environmental Microbiology.

[27]  D. Lilley,et al.  Structures of helical junctions in nucleic acids , 2000, Quarterly Reviews of Biophysics.

[28]  Niels Tolstrup,et al.  OligoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling , 2003, Nucleic Acids Res..

[29]  Gang Bao,et al.  Hybridization kinetics and thermodynamics of molecular beacons. , 2003, Nucleic acids research.