Various chemical modifications to RNA have been incorporated in attempts to improve their pharmacological properties for RNAi interference (RNAi). Recent studies have shown that small interfering RNA (siRNA) containing 2'-fluoro modifications can elicit gene silencing through RNAi. Despite developments in using chemical modifications for increased stability, safety, and efficiency of these therapeutics, they still face challenges of spatial and temporal targeting. One potential targeting strategy is to use photocaging techniques, which involve the covalent attachment of photolabile compounds to the effector nucleic acid species that block bioactivity until exposed to near UV light. In this study we demonstrate that fully 2'-fluorinated nucleic acids (FNAs) can be caged for photoactivated gene silencing in cell culture and in zebrafish embryos. This strategy combines the improvement in chemical and enzymatic stability associated with 2'-substitutions with the targeting ability of a photoinducible trigger. Statistical alkylation of FNAs with 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) improved resistance to enzymatic degradation, reduced RNAi effectiveness, and protected the biological system from toxic doses of the effector. Photo-exposure to 365 nm light partially restored the silencing activity of the 2'-fluoro siRNAs. These results suggest that photocaging may offer control over RNAi therapeutics for spatially and temporally directed activation, while improving enzymatic stability and potentially enabling therapeutic dosing via light dose intensity.

Various strategies have been employed to achieve control over delivery of siRNA molecules to intended target cells. Photocaging is one specific class of modifications for silencing oligonucleotides that block their bioactivity until exposure to near-ultraviolet light. These caged RNAi effectors enable both spatial and temporal targeting of a dosed release of gene silencing agents by directed light exposure that photocleaves the cage moieties. Herein we compare the photochemical properties of cage compounds and strategies for their use, attached either randomly or site-specifically, to demonstrate various forms of gene expression regulation in vitro and in vivo. This light-controllable strategy has potential applications for precisely probing developing biological systems and eventually enabling targeted gene-silencing therapeutics.

RNA interference is a phenomenon in which RNA molecules elicit potent and sequence-specific post-transcriptional gene silencing. Recent studies have shown that small interfering RNA containing pyrimidine 2'-fluoro modifications elicit RNAi. In this study, we demonstrate that fully-2'-fluorinated nucleic acids can be generated for RNAi studies through either custom solid-phase synthesis or in vitro transcription using a mutated polymerase and fluorinated nucleoside triphosphates. Single-stranded and hybridized fully-2'-fluorinated nucleic acids were subjected to a ribonuclease to assess their resistance to digestion. Duplex siFNA and antisense fully-2'-fluorinated nucleic acids were evaluated for their ability to knockdown green fluorescent protein expression in mammalian cell culture. Based on the results, fully-2'-fluorinated nucleic acids can be successfully generated, and fully-2'-fluorinated nucleic acids products show superior resistance to digestion over native RNA. Melt curve analysis suggests that transcribed fully-2'-fluorinated nucleic acids may contain base miscoding errors or early termination products. Small interfering fluoronucleic acid can induce RNAi and the silencing efficiency is nearly equivalent to the unmodified small interfering RNA species. Silencing from antisense fully-2'-fluorinated nucleic acids was greatly reduced relative to the duplex form. The lack of silencing activity from single-stranded fully-2'-fluorinated nucleic acids, combined with reverse transcription polymerase chain reaction data showing that mRNA decreases following siFNA treatment, suggests that knockdown from siFNA is likely enzymatically driven as opposed to simple translational arrest.

Liquid biopsy is a valuable precision oncology tool that is increasingly used as a non-invasive approach to identify biomarkers, detect resistance mutations, monitor disease burden, and identify early recurrence. The Tempus xF liquid biopsy assay is a 105-gene, hybrid-capture, next-generation sequencing (NGS) assay that detects single-nucleotide variants, insertions/deletions, copy number variants, and chromosomal rearrangements. Here, we present extensive validation studies of the xF assay using reference standards, cell lines, and patient samples that establish high sensitivity, specificity, and accuracy in variant detection. The Tempus xF assay is highly concordant with orthogonal methods, including ddPCR, tumor tissue-based NGS assays, and another commercial plasma-based NGS assay. Using matched samples, we developed a dynamic filtering method to account for germline mutations and clonal hematopoiesis, while significantly decreasing the number of false-positive variants reported. Additionally, we calculated accurate circulating tumor fraction estimates (ctFEs) using the Off-Target Tumor Estimation Routine (OTTER) algorithm for targeted-panel sequencing. In a cohort of 1,000 randomly selected cancer patients who underwent xF testing, we found that ctFEs correlated with disease burden and clinical outcomes. These results highlight the potential of serial testing to monitor treatment efficacy and disease course, providing strong support for incorporating liquid biopsy in the management of patients with advanced disease.

We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non–small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97% agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.