US 12,410,463 B2
Molecular beacons
Antony Kuang-Shih Chen, Rockville, MD (US); and Andrew Tsourkas, Philadelphia, PA (US)
Assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, Philadelphia, PA (US)
Appl. No. 13/496,546
Filed by Antony Kuang-Shih Chen, Rockville, MD (US); and Andrew Tsourkas, Philadelphia, PA (US)
PCT Filed Sep. 17, 2010, PCT No. PCT/US2010/049301
§ 371(c)(1), (2), (4) Date May 29, 2012,
PCT Pub. No. WO2011/035135, PCT Pub. Date Mar. 24, 2011.
Claims priority of provisional application 61/243,586, filed on Sep. 18, 2009.
Prior Publication US 2012/0237451 A1, Sep. 20, 2012
Int. Cl. C12Q 1/6876 (2018.01); C12Q 1/6818 (2018.01)
CPC C12Q 1/6818 (2013.01) 10 Claims
 
1. A free-floating bimolecular beacon for nucleic acid detection, comprising:
a first detectable label, a second detectable label, and a quencher;
a first nucleic acid 30 to 100 nucleotides in length; and
a second nucleic acid 10 to 50 nucleotides in length;
wherein said first nucleic acid is longer than said second nucleic acid,
wherein said first nucleic acid forms a stem-loop structure with a loop 10 to 50 nucleotides in length and with a first arm and a second arm, wherein the second arm forms a double-helical region with the first arm by base pairing, wherein the first arm is longer than the second arm and forms an overhanging portion of at least 10 nucleotides, and wherein said loop contains a contiguous sequence complementary to at least 10 consecutive nucleotides of a nucleic acid sequence of interest;
wherein said second nucleic acid is complementary to and forms a double-stranded stem hybrid with the overhanging portion of said first nucleic acid, and wherein the second nucleic acid has an end proximal to the second arm of the first nucleic acid and an end distal to the second arm of the first nucleic acid,
wherein the double-stranded stem hybrid at its end distal from the stem loop has a two base single-stranded 3′-overhang with a sequence of UU,
wherein the second arm of the first nucleic acid is operably linked at its end to the first detectable label or to the quencher,
wherein the second nucleic acid at the end proximal to the second arm of the first nucleic acid is operably linked to the quencher or to the first detectable label, respectively,
wherein the first nucleic acid or the second nucleic acid is operably linked to the second detectable label at an unlabeled end of the first or second nucleic acid, and
wherein the first detectable label is a first fluorophore, the second detectable label is a second fluorophore that is optically distinct from the first fluorophore, and the quencher quenches fluorescence from the first fluorophore by resonance energy transfer.