title: Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East respiratory syndrome coronavirus.
abstract: This paper combines two advances to detect MERS-CoV, the causative agent of Middle East Respiratory Syndrome, that have emerged over the past few years from the new field of "synthetic biology". Both are based on an older concept, where molecular beacons are used as the downstream detection of viral RNA in biological mixtures followed by reverse transcription PCR amplification. The first advance exploits the artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four found in standard DNA and RNA (xNA); AEGIS nucleotides pair orthogonally to the A:T and G:C pairs. Placing AEGIS components in the stems of molecular beacons is shown to lower noise by preventing unwanted stem invasion by adventitious natural xNA. This should improve the signal-to-noise ratio of molecular beacons operating in complex biological mixtures. The second advance introduces a nicking enzyme that allows a single target molecule to activate more than one beacon, allowing "signal amplification". Combining these technologies in primers with components of a self-avoiding molecular recognition system (SAMRS), we detect 50 copies of MERS-CoV RNA in a multiplexed respiratory virus panel by generating fluorescence signal visible to human eye and/or camera.
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, Box 7, Alachua, FL 32615 USA.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Yaren
authors.affiliation: Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, FL 32615, USA.
authors.forename: Lyudmyla G
authors.initials: LG
authors.lastname: Glushakova
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, Box 7, Alachua, FL 32615 USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bradley
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, Box 7, Alachua, FL 32615 USA.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hoshika
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, Box 7, Alachua, FL 32615 USA; Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, FL 32615, USA. Electronic address: manuscripts@ffame.org.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D018352:Coronavirus Infections / Q000175:diagnosis* / Q000821:virology; D005453:Fluorescence; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D059629:Signal-To-Noise Ratio; D014773:Virology / Q000379:methods*
keywords: MERS-CoV; Nicking molecular beacon; RT-PCR; Synthetic biology nucleotides