Compositions and methods for identifying, quantifying, and/or characterizing an analyte
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Publication Number
US-11268135-B2
Publication Date
2022-03-08
Expiration Date
Abstract
Embodiments of this invention are directed towards the sensitive, fast, and accurate identification and/or characterization of a single cell or bacterium, particularly phenotypic characterization. Certain aspects of the invention include assays that include functional nucleic acid probes (FNAPs). FNAPs can be used to generate deoxyribozyme cleavage cascades (DRCC) initiated by activation of a FNAP resulting in a detectable signal from a single cell.
Core Innovation
The invention relates to a nucleic acid probe composition that is activated by target binding to initiate a deoxyribozyme ribonuclease cleavage cascade (DRCC) and generate a detectable fluorescence signal. The composition includes a first functional nucleic acid probe in which quencher or fluorophore labeling is coupled at a first end and a second end, including configurations where the labels are coupled directly or by terminal linkers.
From the first end to the second end, the probe includes a first substrate recognition sequence, a first catalytic core sequence, and a second substrate recognition sequence that is also operable as a landing sequence for an enzymatic sequence, together with a cleavage site cleavable by the enzymatic sequence and a second landing sequence for the enzymatic sequence. The composition further includes a linker comprising a target binding region or a spacer region comprising a target binding region, linking target binding to activation of enzymatic cleavage.
An inhibitory sequence complementary to all or some of the first substrate recognition sequence is included to control probe activation states. When the cleavage site is uncleaved, the first inhibitory sequence, or the first inhibitory sequence together with the second terminal linker, sequesters the first substrate recognition sequence, maintaining a deactivated probe form while cleavage activation allows progression of the cleavage cascade and fluorescent signal generation.
Claims Coverage
Independent claim clm-00001 defines a nucleic acid probe composition with a specific labeled-end architecture and sequence region arrangement, including a cleavage-activated nucleic acid enzyme and an inhibitory sequence that sequesters recognition sequences when cleavage is uncleaved. No other independent claims are explicitly provided in the partial content.
Labeled-end functional nucleic acid probe architecture with quencher and fluorophore labeling
A nucleic acid probe composition comprising a first functional nucleic acid probe having a first end coupled to a quencher or a fluorophore and a second end coupled to a fluorophore or a quencher, with each label coupled directly or by a terminal linker.
Sequence region architecture with catalytic core, enzymatic landing sequences, and cleavage site
A first substrate recognition sequence; a first catalytic core sequence; a second substrate recognition sequence that is also operable as a landing sequence for an enzymatic sequence; a cleavage site that can be cleaved by the enzymatic sequence; and a second landing sequence for the enzymatic sequence.
Target-binding linker or spacer region integrated with enzymatic activation
A linker comprising a target binding region or a spacer region comprising a target binding region, positioned along the sequence between the first and second ends.
Inhibitory sequence sequestering recognition sequence when cleavage site is uncleaved
A first inhibitory sequence that is complementary to all or some of the first substrate recognition sequence, wherein the first inhibitory sequence or the first inhibitory sequence and the second terminal linker sequesters the first substrate recognition sequence when the cleavage site is uncleaved.
The independent claim coverage is centered on a labeled-end functional nucleic acid probe composition that couples target binding to a cleavage-activated nucleic acid enzyme sequence architecture, while using an inhibitory sequence, and optionally a terminal linker, to sequester the recognition sequence in the uncleaved state.
Stated Advantages
Produces a detectable fluorescence signal from single cells or bacteria.
Provides signal initiation via target binding through a deoxyribozyme ribonuclease cleavage cascade (DRCC).
Enables operation without cell lysis.
Supports rapid signal generation in confined or droplet formats.
Supports isothermal operation.
Reduces the need for washing.
Enables multiplexing.
Documented Applications
Single-cell phenotyping by producing detectable fluorescence from single cells.
Detection in bacteria using fluorescence signal generation from bacteria.
Detection workflows using microdroplets/partitions and labeled hairpin/fluorophore-quencher substrates with camera or LED/filter based detection.
Optional readouts for cell viability and environmental stressor readouts, including antimicrobials and chemotherapies.
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