Compositions and methods for the detection of nucleic acids
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Publication Number
US-10876149-B2
Publication Date
2020-12-29
Expiration Date
Abstract
The present invention provides methods for detecting a target nucleic acid in a sample by, for example, incubating the target nucleic acid with a detection probe containing a nucleic acid sequence complementary to at least a portion of the target nucleic acid and a nuclease enzyme that specifically cleaves double-stranded nucleic acids. Hybridization between the detection probe and the target nucleic acid thereby leads to cleavage of the detection probe, releasing a portion of the probe attached to a detectable agent. The portions of the digested probes attached to the detectable agent can be separated from unbound probe and detected in order to determine the presence of the target nucleic acid in the sample. Thus, the invention enables rapid and accurate analysis of a sample for the presence of desired nucleic acid biomarkers.
Core Innovation
The invention describes nucleic-acid region amplification and probe activation in a clinical sample using duplex-specific nuclease (DSN). A nucleic acid detection probe includes a first region, a second region, and a third region, where the second region contains a nucleic acid sequence complementary to at least a portion of a target nucleic acid. Hybridization of the second region to the target nucleic acid forms a double-stranded nucleic acid region that is digested by DSN, and this digestion releases the first region and/or the third region.
The invention further describes repeating digestion and release with additional copies of the nucleic acid probe to amplify the first region and/or the third region. In related embodiments, nucleic acid probe activation is carried out by hybridizing complementary probe regions to a target nucleic acid to form a double-stranded nucleic acid region that is digested by a double-stranded nucleic-acid-selective enzyme. The digestion releases an end region comprising defined probe regions, thereby activating the nucleic acid probe.
The disclosed techniques use duplex-specific nuclease (DSN)-based nucleic acid detection and amplification in which a nucleic acid target hybridizes to nucleic acid detection probes to form a nucleic acid duplex. The DSN selectively digests the double-stranded nucleic acid region of the probe/target complex, releasing labeled probe fragments including released probe end regions. The released labeled probe fragments are separated using immobilized supports such as surfaces or beads and are detected to indicate the presence of target biomarkers.
The disclosed approaches implement duplex-specific amplification (DSA) by repeating DSN cleavage and probe/target hybridization so that released probe regions can be reused to form additional probe/target double-stranded regions. The description provides multiple amplification/detection modes including linear and exponential amplification via release-and-rehybridization, and one-step detection of immobilized probes. Probe designs include RNA block and hairpin probe configurations, and the disclosure includes multiple immobilized probes and single immobilized probes.
Claims Coverage
The independent claims cover three inventive-feature groupings: amplification of a nucleic acid region in a clinical sample, and two methods of activating a nucleic acid probe. Across the claims, the inventive concept centers on hybridizing probe regions to a target nucleic acid to form a double-stranded nucleic acid region and digesting that region with an enzyme capable of selectively digesting double-stranded nucleic acids to release defined probe regions.
Amplifying a nucleic acid region in a clinical sample with DSN digestion and repeating hybridization
A method of amplifying a nucleic acid region in a clinical sample by providing a mixture with a target nucleic acid, a plurality of nucleic acid probes having first, second, and third regions, DSN, and a lysis buffer; hybridizing the second region to the target to form a double-stranded nucleic acid region and digesting the double-stranded region with the DSN to release the first and third regions; and repeating the hybridization/digestion with additional copies of the nucleic acid probe to amplify the first region and/or the third region.
Activating a nucleic acid probe by releasing probe end region after double-stranded selective digestion
A method of activating a nucleic acid probe by providing a clinical sample mixture with a target nucleic acid, a nucleic acid probe having first, second, third, and fourth regions, a double-stranded nucleic acid-selective enzyme, and a lysis buffer; hybridizing the first and second probe regions to the target to form a double-stranded nucleic acid region and digesting at least a portion of the double-stranded region with the enzyme to release an end region comprising the third and fourth regions, thereby activating the nucleic acid probe.
Activating a nucleic acid probe by releasing first and third regions after double-stranded selective digestion
A method of activating a nucleic acid probe by providing a clinical sample mixture with a target nucleic acid, a nucleic acid probe having first, second, and third regions in which the second region comprises a sequence complementary to the target, an enzyme capable of selectively digesting double-stranded nucleic acids, and a lysis buffer; hybridizing the second region to the target to form a probe-target double-stranded nucleic acid region and digesting the double-stranded region with the enzyme to release the first and third regions, thereby activating the nucleic acid probe.
The claims collectively cover DSN- or double-stranded-nucleic-acid-selective enzyme digestion of a probe/target double-stranded region in a clinical sample, with release of defined probe regions used to amplify a nucleic acid region or activate probe function.
Stated Advantages
The system emphasizes robustness based on mismatch specificity and DSN performance in lysis buffer conditions, including activity in sodium dodecyl sulfate (SDS) lysis buffer and protease resistance.
Linear and exponential amplification via release-and-rehybridization is described.
One-step detection of immobilized probes is described.
Documented Applications
Detection of target biomarkers.
Use in clinical sample matrices.
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