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Publication Number
US-12473587-B2
Publication Date
2025-11-18
Expiration Date
Abstract
A method and a kit for enriching target nucleic acid sequences from a biological sample are disclosed. The method includes preparing, and contacting with the biological sample, a first RNA probe set and a second RNA probe set respectively and concurrently targeting both of the two antiparallel strands of a duplex segment in each target nucleic acid sequence. Each RNA probe in the first and second RNA probe set can be generated by chemical synthesis or by in vitro or in vivo transcription, and can be biotin-labelled to thereby allow capturing of the target nucleic acid sequences by magnetic beads labelled with streptavidin, or can be engineered to a microfluidic channel to facilitate the capturing. The method can be applied to capture double-stranded nucleic acid sequences or single-stranded nucleic acid sequences having duplex segments, and the nucleic acid sequences can include DNAs, RNAs, or DNA-RNA hybrid molecules.
Core Innovation
The invention relates to enriching at least one target nucleic acid sequence from a biological sample that contains one or more other sequences. A solid support provides a working surface functionalized with coupling partners to bind immobilization portions of RNA probes. At least one pair of RNA probe sets is provided, where each pair includes a first RNA probe set and a second RNA probe set configured to concurrently and respectively target two antiparallel strands of a duplex segment in each target nucleic acid sequence.
During capturing, the two antiparallel strands of the duplex segment in the target nucleic acid sequence are hybridized concurrently and respectively to the first RNA probe set and the second RNA probe set in each pair. The immobilization portion of each RNA probe binds to the coupling partners on the solid support during or after hybridization, thereby capturing the target nucleic acid sequence on the solid support. The capturing is performed such that the duplex-segment antiparallel strands are targeted at the same time by paired probe sets.
The invention further addresses non-specific binding by blocking non-target sequences prior to or concurrent with hybridization. At least one blocking oligonucleotide is hybridized with the polynucleotide in the biological sample so that the blocking oligonucleotide hybridizes with and blocks one strand in at least one of the one or more other sequences, thereby reducing non-specific binding of the RNA probes.
Claims Coverage
The document contains one independent claim directed to a paired RNA-probe-set enrichment method that concurrently captures two antiparallel strands of a duplex segment using probe immobilization on a solid support, with blocking to reduce non-specific binding.
Paired RNA probe sets concurrently targeting antiparallel strands of a duplex segment
Each pair of RNA probe sets includes a first RNA probe set and a second RNA probe set configured to concurrently and respectively target two antiparallel strands of a duplex segment in each target nucleic acid sequence, where each RNA probe comprises an immobilization portion for attachment to the solid support.
Solid support with coupling partners binding immobilization portions during or after hybridization
The solid support has a working surface functionalized with coupling partners configured to bind immobilization portions of the RNA probes, and the immobilization portion of each RNA probe binds to the coupling partners on the solid support during or after hybridization to capture the target nucleic acid sequence on the solid support.
Blocking non-target sequences to reduce non-specific binding of RNA probes
Prior to or concurrent with hybridization, at least one blocking oligonucleotide is hybridized with the polynucleotide in the biological sample such that the blocking oligonucleotide hybridizes with and blocks one strand in at least one of the one or more other sequences, and sterically or competitively inhibits hybridization of the RNA probes to non-target sequences to reduce non-specific binding.
Pre-immobilized paired RNA probe sets for duplex-strand capturing
For each pair of RNA probe sets, the method includes pre-immobilizing the RNA probe sets on the solid support working surface so that during the capturing step the immobilized first and second RNA probe sets hybridize to the two antiparallel strands of the duplex segment.
Physically separated immobilized positions for probes of each RNA probe pair
For each pair of RNA probe sets, all probes in the first and second RNA probe sets are immobilized at physically separated positions on the working surface of a solid support.
Sequential hybridization reactions contacting first and second RNA probe sets
The method contacts a first and a second RNA probe set with target nucleic acid sequences sequentially using a first hybridization reaction followed by a second hybridization reaction for each RNA probe set pair.
Solid support working surface options functionalizable with coupling partners
A working surface is selected from specified surface types such that a solid support on that working surface can be labeled, conjugated, or attached with an appropriate coupling partner for immobilization.
Overall claim coverage centers on concurrently hybridizing a duplex segment’s two antiparallel strands using paired first and second RNA probe sets immobilized via immobilization portions to coupling partners on a solid support, with blocking oligonucleotides that sterically or competitively inhibit non-target binding.
Stated Advantages
Higher capture efficiency versus single-strand capture, as asserted in the document.
Improved SNV accuracy using complementary-strand matching, as asserted in the document.
Ultra-rare mutation detection performance, as asserted in the document.
Documented Applications
Downstream use for NGS and PCR-based analyses, including SNV calling, CNV analysis, and structural variant detection, as described.
Whole exome sequencing (WES) and barcoded single-stranded library analysis with unique read families (URFs) and super reads, as described.
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