Molecular beacons
Inventors
Chen, Antony Kuang-Shih • Tsourkas, Andrew
Assignees
University of Pennsylvania Penn
Publication Number
US-12410463-B2
Publication Date
2025-09-09
Expiration Date
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Abstract
The invention provides novel compositions and methods for the detection of a target molecule. Specifically, the invention provides a bimolecular beacon composition comprising two nucleic acid molecules, and methods thereof. One of the nucleic acid molecule is operably linked to a reporter detectable label and the other nucleic acid molecule is operably linked to a reference detectable label and a quencher. In some embodiments, at least one of the nucleic acid molecules comprises a single stranded overhang.
Core Innovation
The invention provides novel compositions and methods for the detection of a target molecule. Specifically, the invention provides a bimolecular beacon comprising two nucleic acid molecules, wherein one nucleic acid molecule is operably linked to a reporter detectable label and the other nucleic acid molecule is operably linked to a reference detectable label and a quencher. In some embodiments, at least one of the nucleic acid molecules comprises a single stranded overhang. The bimolecular beacon is capable of emitting both reporter and reference fluorescence with the same probe, thereby accounting for cell-to-cell variability and allowing confident analysis of gene expression in individual cells.
The background identifies problems with conventional molecular beacons, including rapid sequestration into the nucleus that elicits a bright false-positive signal, non-specific opening leading to ambiguous results and loss in sensitivity and dynamic range, and inability to provide accurate information on cell-to-cell variations in gene expression owing to heterogeneous delivery. The invention addresses these problems by providing a bimolecular beacon design with a long double-stranded domain and an overhang that facilitates nuclear export, and an unquenched reference fluorophore to monitor probe delivery and enable ratiometric quantification of probe hybridization.
Claims Coverage
The patent includes two independent claims and the following main inventive features are identified from those claims.
Bimolecular beacon structure
A free-floating bimolecular beacon for nucleic acid detection comprising a first detectable label, a second detectable label, and a quencher.
First and second nucleic acid lengths
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.
Stem-loop and loop complementarity
The 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 loop contains a contiguous sequence complementary to at least 10 consecutive nucleotides of a nucleic acid sequence of interest.
Asymmetric arm and overhanging portion
The first arm is longer than the second arm and forms an overhanging portion of at least 10 nucleotides, and the second nucleic acid is complementary to and forms a double-stranded stem hybrid with the overhanging portion of the first nucleic acid.
UU three-prime overhang on stem hybrid
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.
Operable linkage of labels and quencher
The second arm of the first nucleic acid is operably linked at its end to the first detectable label or to the quencher, and 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; and the first nucleic acid or the second nucleic acid is operably linked to the second detectable label at an unlabeled end.
Distinct fluorophores and quenching mechanism
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.
Single detectable label embodiment
An alternative free-floating bimolecular beacon embodiment comprising a detectable label and a quencher, with the first and second nucleic acids and structural features as recited, wherein the detectable label is a fluorophore and the quencher quenches fluorescence from the fluorophore by resonance energy transfer.
The independent claims define a bimolecular beacon composed of two hybridized nucleic acids of specified lengths and asymmetric stem-loop architecture with a UU 3′-overhang, specific operable linkages of fluorophore(s) and quencher, and a resonance energy transfer quenching mechanism; one independent claim recites embodiments with two optically distinct fluorophores and the other recites an embodiment with a single detectable fluorophore.
Stated Advantages
Emits both reporter and reference fluorescence with the same probe, thereby accounting for cell-to-cell variability and allowing confident analysis of gene expression in individual cells.
Allows monitoring of transfection efficiency and unbound probe localization through an unquenched reference fluorophore, reducing false-negatives and enabling differentiation between untransfected cells and low gene expression.
Facilitates nuclear export by inclusion of a long double-stranded domain with a single-stranded overhang, thereby avoiding nuclear sequestration and related false-positive signals.
Enables ratiometric imaging to quantify probe hybridization and improve detection sensitivity by normalization, removing instrumental and experimental variability.
Provides improved sensitivity for RNA detection in living cells, high resistance to nonspecific opening, and prolonged functional activity in cells compared with conventional molecular beacons.
Documented Applications
A method for detecting a polymorphism associated with a target gene by providing the molecular beacon, detecting first and second detectable labels, and determining a relative fluorescent intensity of the labels.
A method for detecting the expression level of a target gene by providing the molecular beacon, detecting first and second detectable labels, and determining a relative fluorescent intensity of the labels.
A method for diagnosis of a disease in a subject by providing the molecular beacon, detecting first and second detectable labels, and determining a relative fluorescent intensity of the labels to indicate presence or absence of disease.
A method for monitoring a transfection efficiency by providing the molecular beacon, detecting first and second detectable labels, and determining a fluorescent intensity of the labels to indicate transfection efficiency.
Nucleic acid detection in vitro and in living cells and use as part of a genomic marker test kit for identifying cells which express a particular protein by measuring protein-encoding nucleic acid levels in a sample.
Visualization of the location and relative amount of gene expression in tissues and cells through fluorescence or luminescence, including in vivo imaging in living cells and tissues.
Use in amplification reactions as primers or, in triamplification, as blocking oligonucleotides to detect or measure nucleic acid products and thereby detect or measure target nucleic acids in a sample.
A screening tool for drug discovery to detect in vivo changes in expression of transcripts of interest and to monitor dose-dependent cellular responses to external stimuli.
Kits for detection comprising the nucleic acid probe compositions, reagents, instructions, and optionally standards and software for correlating values and storing results.
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