Characterization of mRNA sequences is a critical aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can positively identify a single correct sequence from hundreds of similarly sized sequences. In addition, the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.

Eluforsen (previously known as QR-010) is a 33-mer 2′- O -methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3′ exonuclease were observed in mouse liver in an in vitro incubation system and by either 3′ exonuclease or 5′ exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2′-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics.

Eye-tracking provides an opportunity to generate and analyze high-density data relevant to understanding cognition. However, while events in the real world are often dynamic, eye-tracking paradigms are typically limited to assessing gaze toward static objects. In this study, we propose a generative framework, based on a hidden Markov model (HMM), for using eye-tracking data to analyze behavior in the context of multiple moving objects of interest. We apply this framework to analyze data from a recent visual object tracking task paradigm, TrackIt, for studying selective sustained attention in children. Within this paradigm, we present two validation experiments to show that the HMM provides a viable approach to studying eye-tracking data with moving stimuli, and to illustrate the benefits of the HMM approach over some more naive possible approaches. The first experiment utilizes a novel 'supervised' variant of TrackIt, while the second compares directly with judgments made by human coders using data from the original TrackIt task. Our results suggest that the HMM-based method provides a robust analysis of eye-tracking data with moving stimuli, both for adults and for children as young as 3.5-6 years old.

Quantitative bioanalysis in plasma and tissues samples is required to study the pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides (ASOs). To overcome intrinsic drawbacks in specificity, sensitivity, and throughput of traditional ligand-binding assay (LBA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods, an alternative bioanalytical method was developed by combining oligonucleotide hybridization and LC–MS/MS technologies. Target ASOs were extracted from biological samples by hybridization with biotinylated sense-strand oligonucleotides coupled to streptavidin magnetic beads. Using ion-pairing chromatography and tandem mass spectrometry, this method demonstrated high sensitivity (0.5 ng/mL using 100 μL of plasma), high specificity, wide linear range, complete automation, and generic applications in tests with multiple ASOs. The typical challenge of sensitivity drop in traditional ion-pairing LC–MS/MS was for the first time overcome by the introduction of a ternary pump system. Due to the high specificity, quantitation in various biological matrixes was achieved using calibration standards in plasma, largely improving efficiency and consistency. Another major advantage was the capability of simultaneous quantitation of ASO metabolites. The hybridization LC–MS/MS was considered an improved alternative for quantitation of ASOs and metabolites in plasma and tissue samples, showing a great potential to replace traditional LBA and LC–MS/MS methods.

The high prevalence and long latency period of prostate cancer (PCa) provide a unique opportunity to control disease progression with dietary and nutraceutical approaches. We developed ProFine, a standardized composition of luteolin, quercetin, and kaempferol, and investigated its potential as a nutraceutical for PCa in preclinical models. The three ingredients of ProFine demonstrated synergistic in vitro cytotoxicity and effectively induced apoptosis in PCa cells. ProFine markedly affected the transcriptome of PCa cells, suppressed the expression of androgen receptor, and inhibited androgen-regulated genes. Oral administration of ProFine did not exhibit obvious toxicities in mice, and the three ingredients retained their individual pharmacokinetic and bioavailability profiles. Importantly, ProFine significantly retarded the growth of PCa xenografts in athymic nude mice and extended the survival of animals. This study provides preclinical evidence supporting the promise of ProFine as a safe, efficacious, and affordable intervention to control PCa progression and improve clinical outcomes.