Time-resolved nucleic acid hybridization probes
Inventors
Medintz, Igor L. • Ancona, Mario • Algar, W. Russ • Massey, Melissa M.
Assignees
Publication Number
US-11359230-B2
Publication Date
2022-06-14
Expiration Date
2037-03-24
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Abstract
Time-resolved nucleic acids include a long-lifetime FRET donor with an emission lifetime of at least one millisecond (such as a terbium complex), configured as a donor in a first FRET process, and at least one fluorescent dye with an emission lifetime of less than 100 nanoseconds configured as an acceptor in the FRET process. They can be configured as photonic wires, hybridization probes or beacons, and/or systems for computing logical operations.
Core Innovation
This invention provides nucleic acid hybridization probes employing a long-lifetime FRET donor with an emission lifetime of at least one millisecond, such as a terbium complex, combined with at least one fluorescent dye acceptor having an emission lifetime less than 100 nanoseconds. These probes are constructed such that the donor and acceptor dyes are arranged to optimize FRET efficiency within a “sweet spot” distance that balances energy transfer efficiency and temporal gating requirements. The probes can be designed as photonic wires, hybridization probes or beacons, and systems capable of computing logical operations using nucleic acids.
The problem addressed is improving the sensitivity and selectivity of nucleic acid detection and biological material assays using fluorescence methods. Conventional fluorescent dyes suffer limitations including photobleaching, non-optimal orientation factors in FRET, and background fluorescence interference from sample matrices or excess dye-labeled strands. These factors reduce signal-to-background ratios and complicate detection. The invention solves these issues by utilizing a luminescent terbium complex as a long-lifetime FRET donor, enabling time-gated fluorescence detection that minimizes background and enhances assay performance.
The invention demonstrates that positioning the long-lifetime donor and fluorescent acceptor at a specific distance—the sweet spot—enables effective time-gated Förster resonance energy transfer (FRET) cascades in DNA-based constructs. This spatial and temporal optimization overcomes premature quenching and signal loss associated with very high or very low FRET efficiencies. Including locked nucleic acids (LNA) in the scaffolds increases thermal stability of the photonic structures, improving operational robustness. The system configurations enable multiplexed detection, selective hybridization target sensing, and the construction of nucleic acid logic operators that process molecular inputs into photonic outputs.
Claims Coverage
The patent contains one independent claim, covering a nucleic acid hybridization probe with defined structural and photophysical features. The main inventive features pertain to probe composition, configuration of FRET elements, and assay applications.
Nucleic acid hybridization probe with long-lifetime luminophore and fluorescent dye acceptor
A probe oligonucleotide 15 to 21 nucleotides in length, covalently attached at one end to a long-lifetime luminophore (emission lifetime ≥1 ms) configured as a FRET donor, and at the opposite end to a fluorescent dye with emission lifetime <100 ns configured as a FRET acceptor. The donor and acceptor are positioned at a "sweet spot" distance that balances FRET efficiency and time-gated detection lag time.
Blocked probe configuration with complementary blocker oligonucleotide exposing a toehold
The probe further includes a blocker oligonucleotide complementary and paired to the probe, shorter by four to six nucleotides, hybridized at the end with the luminophore to expose a toehold region. The blocker carries a quencher that efficiently quenches fluorescence emission from the long-lifetime luminophore, thereby modulating the FRET signal depending on target presence.
Specific probe and blocker length and donor composition
In an embodiment, the probe oligonucleotide comprises 18 nucleotides, the blocker has 12 nucleotides, and the long-lifetime FRET donor is a lanthanide complex containing terbium(III) or europium(III).
Multiplexed probe sets with different sequences and reporter dyes
Multiple probes having different nucleotide sequences are combined in a set, each sequence variant labeled with a different fluorescent dye acceptor, enabling multiplexed detection of various targets.
Method of nucleic acid target detection using the hybridization probes
Methods involve contacting a sample containing a nucleic acid target with the described probe, where the probe sequence is complementary to the target, and measuring time-gated fluorescence intensity of the fluorescent dye, where the intensity correlates with target concentration.
Detection in complex biological matrices
The detection methods are effective in samples comprising up to 90% serum or 90% blood by volume, enabling sensing in complex biological fluids.
The claims cover nucleic acid hybridization probes structurally defined by a long-lifetime FRET donor and a short-lifetime fluorescent dye acceptor arranged for optimized time-gated FRET signal, with optional blocking oligonucleotides exposing toeholds for strand displacement signaling. Methods for detecting nucleic acid targets using these probes, including in complex biological matrices and multiplexed formats, are included.
Stated Advantages
Minimization of unwanted background fluorescence through time-gated emission measurements enabled by the long-lifetime terbium donor.
Improved signal-to-background ratios by suppressing direct excitation of acceptor dyes and background from excess dye-labeled strands or sample matrix autofluorescence.
Multiplexed detection capability by pairing terbium donor emission lines with different fluorescent acceptor dyes.
Increased thermal stability of photonic wire structures by inclusion of locked nucleic acids (LNA).
Sensitive detection with nanomolar limits of detection suitable for assays in complex matrices such as serum and blood.
Temporal signaling mechanism using a 'sweet spot' donor-acceptor distance that optimizes FRET efficiency with instrument lag time for enhanced assay performance.
Capability to design DNA-based logic operators that combine molecular inputs into photonic outputs, enabling molecular computation.
Documented Applications
Nucleic acid hybridization assays for detecting specific DNA sequences, including those relevant to drug-resistant tuberculosis genetic markers.
Multiplexed nucleic acid detection using probe sets labeled with different fluorescent dyes.
Detection of nucleic acid targets in complex biological sample matrices such as serum and blood.
Time-resolved nucleic acid hybridization beacons employing toehold-mediated strand displacement for target detection.
Unblocked nucleic acid beacon probes relying on intrinsic secondary structure and temporal signaling mechanisms for target detection.
DNA-based photonic logic operators performing Boolean AND, OR, NAND, and NOR logic functions applied to oligonucleotide inputs with photonic fluorescence outputs.
Construction of functional molecular logic device circuits combining multiple DNA logic operators for multiplexed analysis and molecular computation.
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