Reversible fluorescence quenching label design for sensing unlabelled oligonucleotides


Compared with the common approach that employs an oligonucleotide probe with a covalently linked fluorophore, the use of a fluorescently labelled universal reporter strand hybridized to an unlabelled stem-loop probe provides a more efficient approach to the fabrication of nucleic acid sensors and microarrays potentially useful for real-time analysis.

Technology Summary
This system utilizes a stem-loop probe hybridized to a fluorescently labelled universal reporter for sensing unlabelled nucleic acids. At room temperature, the probe and reporter are in the hairpin-closed form in which the fluorophore of the reporter is in proximity to the G bases of the hairpin, where consequently the fluorescent emission is quenched significantly. On hybridization with target, probe and reporter are trapped in the hairpin-opened configuration in which the fluorophore and the G quenchers are apart. This turns off quenching, increases emission intensity, and signals the presence of target.

Features and benefits

  • use of unlabelled sequence-specific probes increases the yield and reduces the cost of the probe
  • use of a single sequence of fluorescently labelled universal reporter for all targets, instead of covalently linking the fluorophore and quencher to each probe sequence, allows efficient synthesis of the reporter in bulk quantities
  • self-assembly (via hybridization) of the sensor motif simplifies the manufacturing process
    the address-binding region allows immobilization by hybridization to specific address sites of microarrays without the need to modify the probe with an attachment group
  • all sensing components can be affixed to a solid substrate where no washing steps are required in the detection of unlabelled targets
  • RNA purification and reverse transcription of cDNA not required
  • the fluorescence quenching output is reversible
  • together these properties minimize the time, cost, and error inherent in the analysis and also allow in vivo or real-time in situ monitoring

Available for non-exclusive or exclusive licensing and UNR is seeking parties interested in collaborative research to further develop, evaluate, or commercialize this technology.

IP Status
UNR ID: UNR04-015
Methods for Detecting and Measuring Specific Nucleic Acid Sequences
US Patent Appl. No.: 10/578,248

Patent Information:
Life Sciences
For Information, Contact:
Dan Langford
Technology Commercialization, Manager
University of Nevada, Reno and Desert Research Institute
Suk-Wah Tam-Chang
Kenneth Hunter
Nelson Publicover