Compositions, methods and devices comprising stem-loop captor molecules
Inventors
KOELLE, Paula M. • CHITTUR, Krishnan • Korman, Valentin • McGEE, Zachary
Assignees
Publication Number
US-11713482-B2
Publication Date
2023-08-01
Expiration Date
2037-06-15
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Abstract
Disclosed herein are methods, devices and compositions comprising nucleic acid captor molecules with a stem region and a loop region for detecting target nucleic acids.
Core Innovation
The present disclosure relates to compositions, methods, devices, kits, and systems for rapidly and sensitively detecting one or more target nucleic acid sequences in environmental or biological samples. These methods utilize nucleic acid captor molecules that have a stem region and a loop region forming a closed stem-loop (hairpin) structure. Upon binding to a complementary target nucleic acid sequence within a sample, the captor molecule changes from a closed to an open stem-loop conformation, exposing the stem region which then binds to a labeled nucleic acid probe. The labeled probe’s binding facilitates detection of the target nucleic acid presence.
The method addresses the limitations of current nucleic acid detection techniques which mostly rely on amplification processes requiring specialized instrumentation, temperature-sensitive reagents, and laboratory environments not suitable for rapid field diagnostics. The disclosed approach allows sensitive and specific detection without the need for target nucleic acid amplification or high-temperature melting conditions that are typical in prior art methods. The captor-probe approach provides single-nucleotide polymorphism discrimination and improves specificity by maintaining the closed stem-loop structure during the initial hybridization, thereby favoring binding only to fully complementary target sequences.
Disclosed devices include substrates, such as microarrays or beads, with captor molecules attached via linkers and spaced apart to prevent formation of captor molecule-dimers that could reduce assay specificity. The method incorporates optimized hybridization buffers containing ionic surfactants, ethanol, or dimethyl sulfoxide to expedite binding and increase specificity. Kits comprising captor molecules, labeled probes, buffers, rinsing solutions, and instructions are provided to facilitate the performance of these rapid nucleic acid detection assays in types of environments unsuitable for traditional nucleic acid amplification methods.
Claims Coverage
The patent claims encompass methods, compositions, devices, systems, and kits related to rapid target nucleic acid detection using captor molecules with stem-loop structures and labeled probes, emphasizing spacing, buffering, and controls.
Method for detecting target nucleic acids using spaced captor molecules and negative controls
A method comprising contacting target nucleic acids to captor molecules attached to a substrate by linkers where captors are spaced apart to prevent captor molecule-dimers and include one or more negative controls attached to the substrate.
Captor molecule spacing to prevent dimer formation
Captor molecules are spaced apart by at least half of the length of their closed hairpin structure to inhibit captor molecule-dimer formation.
Use of a specific negative control captor molecule
Inclusion of a negative control captor molecule comprising SEQ ID NO: 160 to monitor specificity and background binding in the assay.
Pre-concentration of target nucleic acids
Prior to contacting captor molecules, target nucleic acids may be concentrated to enhance assay sensitivity.
Use of ascorbic acid in rinsing
After probe binding, rinsing includes adding a solution with ascorbic acid to enhance fluorescence detection and removal of unbound probe.
Hybridization buffering conditions for rapid and specific binding
Buffers comprising ionic surfactants such as sodium dodecyl sulfate (0.005%-0.2% v/v), ethanol (5%-30% v/v), dimethyl sulfoxide (0.10 M to 1.0 M), or combinations thereof to facilitate rapid and specific hybridization.
Shorter probe length relative to stem region
Detectable probes contain fewer nucleotides complementary to the captor stem region than the total nucleotides in the captor stem to reduce non-specific probe binding.
Assay device with competitive binding inhibitors
The assay device includes competitive binding inhibitors attached to the substrate to decrease non-specific interactions.
Competitive binding inhibitor comprising a linker and specific nucleic acid sequence
Competitive binding inhibitors comprise a linker attached to SEQ ID NO: 30.
Use of specific captor and probe sequences in detection
Captor molecule comprising SEQ ID NO: 1 and probe comprising SEQ ID NO: 2 are employed in the detection method.
Compositions of detectable probes for the method
Compositions include detectable probes selected from SEQ ID NOs: 2, 7, 16, 24, and 336-338.
Use of helper oligos to improve target binding
Helper oligos selected from SEQ ID NOs: 324-335 are employed to enhance target nucleic acid accessibility and binding.
Wide range of captor molecules for detection
Captor molecules selected from SEQ ID NOs: 1, 3-6, 8, 15, 17, 19, 21-22, 25, 27, 29, 32-323, and 339 are used in assays to detect various targets.
Assay device comprising substrate with spaced captor and negative control molecules
A device that includes captor molecules attached via linkers spaced to prevent dimerization and one or more negative control captor molecules attached to the substrate.
Incorporation of binding inhibitors in assay device
The device further comprises binding inhibitors attached to the substrate to manage non-specific binding.
Inclusion of specific negative control captor molecules
Specific negative control captor molecules are attached to the assay substrate to serve as refined controls.
System comprising assay device, buffers, and probes
A system that includes an assay device with spaced captors and negative controls, solutions comprising buffers or rinses, and one or more detectable nucleic acid probes.
System with assay device comprising competitive binding inhibitors
The assay device substrate further includes attached competitive binding inhibitors for improved assay performance.
Kit comprising captor molecules and labeled probes
A kit including nucleic acid captor molecules with closed stem-loop structures that open upon target binding, and labeled probes that bind to the exposed stem region of the open captor molecules.
The claims define innovations in nucleic acid detection employing stem-loop captor molecules attached to substrates at controlled spacing, negative control captors, competitive inhibitors, optimized hybridization conditions, and labeled probes shorter than the captor stem region, collectively enabling rapid, sensitive, and specific detection of target nucleic acids in various assay formats and devices.
Stated Advantages
Provides rapid and sensitive detection of nucleic acids without the need for target amplification or high temperature melting.
Allows for single nucleotide polymorphism (SNP) discrimination, increasing specificity in target detection.
Enables nucleic acid detection in field and conflict settings without requiring laboratory infrastructure or temperature sensitive reagents.
Reduces background noise by using labeled probes that bind only to open stem-loop captors having hybridized target nucleic acids, allowing for effective rinsing of unbound probes.
Facilitates multiplexed detection of multiple target nucleic acids simultaneously on arrays or bead-based platforms.
Uses hybridization buffers with ionic surfactants, ethanol, or DMSO to shorten assay time while improving signal and specificity.
Improves assay performance by spacing captor molecules to prevent formation of captor molecule dimers that increase non-specific probe binding.
Incorporates competitive binding inhibitors and specific negative controls to reduce non-specific interactions and enhance assay accuracy.
Documented Applications
Rapid detection and quantification of nucleic acids for identifying organisms or viruses in biological and environmental samples.
Monitoring gene expression levels or alterations in response to therapeutic compounds or stimuli.
Testing bacterial susceptibility to antibiotics by measuring changes in target nucleic acid levels after exposure to antibiotics.
Field-deployable diagnostics in epidemic, pandemic, or battlefield settings lacking laboratory facilities.
Simultaneous detection of multiple nucleic acids in a single sample using microarray or bead-based assay platforms.
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