Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE.

BACKGROUND: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed. RESULTS: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run. CONCLUSION: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.

Single-copy detection of somatic variants from solid and liquid biopsy.

Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

CDK12 inhibition reduces abnormalities in cells from patients with myotonic dystrophy and in a mouse model.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

Synthesis of triazole-linked morpholino oligonucleotides via Cu(I) catalysed cycloaddition.

Triazole-linked morpholino ((TL)MO) oligonucleic acids were synthesised using the Cu(I) catalysed (3 + 2) azide-alkyne cycloaddition (CuAAC) reaction. The modified DNA analogues were incorporated into 13-mer sequences via solid phase synthesis. UV melting experiments showed that the (TL)MO modification gives higher Tm values than the corresponding (TL)DNA modification.