Time-resolved nucleic acid hybridization beacons
Inventors
Medintz, Igor L. • Ancona, Mario • Algar, W. Russ • Massey, Melissa M.
Assignees
Publication Number
US-10465233-B2
Publication Date
2019-11-05
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
The invention pertains to the development of time-resolved nucleic acid systems utilizing a long-lifetime FRET donor, such as a luminescent terbium complex with an emission lifetime of at least one millisecond, configured as a donor in a first FRET process and paired with fluorescent dyes having emission lifetimes of less than 100 nanoseconds as acceptors in the FRET process. These systems are embodied as photonic wires, nucleic acid hybridization probes or beacons, and systems for computing logical operations. The photonic wires leverage DNA or DNA/LNA scaffolds to arrange dyes at precise distances facilitating FRET cascades, where the long donor lifetime permits time-gated fluorescence measurements minimizing background signals.
The problem addressed arises from limitations in conventional DNA-organized FRET-based photonic wires, including dye photobleaching, undesirable direct excitation of downstream dyes, suboptimal donor-acceptor orientations, lower than predicted transfer efficiencies, and sensitivity to incomplete nanostructure formation. Additionally, traditional fluorescent assays face challenges from background fluorescence in complex biological samples and restricted multiplexing. The invention solves these challenges by employing luminescent lanthanide complexes as long-lifetime donors enabling time-gated measurements that reject short-lifetime background fluorescence, improving signal-to-noise ratios and facilitating multiplexed detection in complex sample matrices.
Furthermore, the invention introduces the concept of a 'sweet spot' for donor-acceptor spacing in time-gated FRET systems, balancing FRET efficiency and temporal detection windows to maximize time-gated emission signal. This insight enables optimization of probe designs for improved assay sensitivity and selectivity. The detailed design of hybridization probes includes blocked and unblocked beacon constructs labeled with lanthanide donors and fluorescent acceptors, exploiting time-gated FRET and strand displacement mechanisms for target detection with nanomolar sensitivity and multiplexing capability, even in complex biological matrices.
Claims Coverage
The patent includes multiple independent claims covering photonic wire constructs featuring nucleic acid chains labeled with long-lifetime FRET donors and fluorescent dye acceptors configured to achieve optimized FRET efficiency at defined distances. Key inventive features from these claims are summarized below.
Photonic wire design with a long-lifetime FRET donor and fluorescent dye acceptors configured at a sweet spot distance
A photonic wire comprising: a first nucleic acid chain including a long-lifetime FRET donor with an emission lifetime of at least one millisecond serving as a donor in a first FRET process; a second nucleic acid chain complementary to and paired with the first, comprising a first fluorescent dye that acts as a FRET acceptor in the first process and a donor in a second FRET process. The second chain and first nucleic acid chain position the donor and first fluorescent dye at a sweet spot distance between about 0.25 to 1.0 times the predicted Förster distance. A third nucleic acid chain, also complementary and paired adjacent to the second, comprises a second fluorescent dye configured as an acceptor in the second FRET process. Each fluorescent dye has an emission lifetime of less than 100 nanoseconds, and the sweet spot distance balances FRET efficiency and lag time.
Use of lanthanide complexes as long-lifetime FRET donors
The photonic wire includes a long-lifetime FRET donor comprising a lanthanide complex selected from terbium(III) or europium(III) complexes.
Incorporation of locked nucleic acid components for enhanced stability
At least one nucleic acid chain in the photonic wire includes locked nucleic acid residues to increase thermal stability of the construct.
Extension of photonic wire with additional fluorophore-labeled nucleic acid chains
The photonic wire may further include additional nucleic acid chains complementary to the first nucleic acid chain and paired adjacent to existing chains, each comprising additional fluorescent dyes configured as FRET acceptors.
The independent claims cover photonic wire constructs leveraging long-lifetime lanthanide FRET donors paired with short-lifetime fluorescent dye acceptors arranged at optimized 'sweet spot' distances along nucleic acid scaffolds, optionally incorporating locked nucleic acid bases and extended with multiple donor-acceptor chains, collectively enabling enhanced time-resolved FRET with balanced efficiency and temporal resolution.
Stated Advantages
Minimization of unwanted background fluorescence from direct dye excitation and sample matrix autofluorescence via time-gated fluorescence measurements.
Improved signal-to-background ratios in FRET assays through the use of long-lifetime lanthanide donors and optimized donor-acceptor spacing.
Capability for multiplexed detection enabled by multiple emission lines of the terbium donor and varied fluorescent acceptors.
Increased thermal stability of DNA nanostructures incorporating locked nucleic acid residues.
Enhanced selectivity and sensitivity in nucleic acid hybridization probes and beacons through competitive energy transfer and toehold-mediated strand displacement.
Suitability for detection in complex biological samples such as serum and blood by reducing autofluorescence and background interference.
Provision of novel temporal signaling mechanisms in TR-FRET assays exploiting the 'sweet spot' for donor-acceptor spacing.
Functional DNA-based photonic logic gates capable of computable molecular logic operations with photonic outputs responsive to nucleic acid inputs.
Documented Applications
Design and application of DNA/LNA photonic wires for efficient, time-gated FRET cascades facilitating nanoscale photonic energy transfer.
Development of time-gated nucleic acid hybridization probes or beacons for sensitive and selective detection of nucleic acid targets in biomedical diagnostics.
Multiplexed detection of different DNA sequences via probes labeled with fluorescent dyes emitting at distinguished wavelengths.
Nucleic acid hybridization assays operational within complex biological matrices including serum and blood.
Construction and operation of DNA-based photonic logic operators (AND, OR, NAND, NOR gates) performing logical computations via time-gated FRET signaling responsive to nucleic acid input sequences.
Prospective applications in biosensing, biophotonic logic, molecular diagnostics, and molecular computation using DNA nanotechnology.
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