SIGNIFICANCE STATEMENT: A novel hybridization LC-MS/MS-based approach was successfully developed for the determination of ASO in vitro protein binding in plasma, and for the first time brain and CSF. As ASOs continue to undergo clinical trials for neurologic and neuromuscular indications, ƒ u characterization in brain and CSF can provide invaluable information about ASO distribution and target engagement in the central nervous system, therefore providing support for in vivo PK/PD data characterization. Although ASO protein binding has been previously characterized in plasma, there were no studies that quantitated ASO ƒ u in brain or cerebral spinal fluid (CSF). The hybridization LC-MS/MS platform was integrated with ultracentrifugation, ultrafiltration, and equilibrium dialysis, and method performance for each technique was evaluated. The current study describes the implementation of a hybridization liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for the direct quantitation of antisense oligonucleotide (ASO) ƒ u The method provides substantial improvements, including minimal matrix effects and high specificity when compared with previously used oligonucleotide ƒ u detection methods such as ligand binding assays or liquid scintillation. Drug unbound fraction (ƒ u) characterization is a key consideration in pharmacokinetic and pharmacodynamic (PK/PD) modeling, assuming only unbound drug can interact with the target, and therefore has direct implications in the efficacy and potential toxicity of the drug. RT-PCR detection of ACoS-AS1 in cDNA isolated from PI 114490 (PI), six red-fruited tomato lines Huangrong Red (HGR), TS-196, TS-223, Zhengyang (ZY), Jinjue (JJ), and OH 88119), and one pink-fruited tomato line TS-35.The development of quantitative models for prediction of drug pharmacokinetics based on in vitro data has transformed early drug discovery. Variants 1 (V1), 2 (V2) and 3 (V3) are amplified with primer pairs PSY1F1/PSY1U-R4, PSY1F1/PSY1U-R9, and PSY1F1/PSY1U-R13, respectively. Image of agarose gel for PCR products of three variants amplified from cDNA of PI 114490. Variant 3 is the possible real transcript of ACoS-AS1, while variants 1 and 2 are alternative transcripts by retaining part of the second intron. Empty boxes represent potential exons, black line represents the first intron, and grey, blue, pink and green boxes represent the potential second intron. Diagram of three transcripts and their origin in the genomic sequence. Primers are designed based on the reverse sequence. Black box indicates exon of SlPsy1 and acyl-CoA synthase gene, while black line indicates intron and intergenic region. Red triangle indicates primers with the success of PCR amplification. Positions of 15 primers (Table S1) for detecting transcript variants on the opposite DNA sequence of SlPsy1 gene region. Three transcripts of ACoS-AS1 in yellow-fruited tomato line PI 114490. SlPsy1 Trans-splicing carotenoids lncRNA tomato yellow fruit. The results obtained here will extend knowledge to understand the mechanism of trans-splicing event SlPsy1-ACoS-AS1 and provide additional information for the regulation of carotenoids biosynthesis. Mutation of ACoS-AS1 in PI 114490 generated by CRISPR/Cas9 techniques resulted in red fruits implying that ACoS-AS1 was essential to trans-splicing event SlPsy1-ACoS-AS1. Sub-cellular localization analysis showed that SlPSY1-ACoS-AS1 could not enter plastids where SlPSY1 has its enzyme activity. Over-expression of SlPsy1-ACoS-AS1 in red-fruited tomato line M82 did not have any phenotype change while over-expression of wild type SlPsy1 resulted in altered leaf colour. However, transgenic tomato lines carrying the genomic DNA of SlPsy1 from PI 114490 did not generate transcripts of ACoS-AS1and SlPsy1-ACoS-AS1 suggesting that only the intronic SNP could not cause the trans-splicing event. The intronic SNP and intergenic SSR were tightly associated with trans-splicing event SlPsy1-ACoS-AS1. The data showed that the previously defined unknown gene was a putative long non-coding RNA ACoS-AS1 with three variants in many yellow-fruited tomato lines. In the current study, the cause of trans-splicing event was further investigated. The genomic DNA sequences of SlPsy1 between red and yellow-fruited tomato lines have one single-nucleotide polymorphism (SNP) in the fourth intron and one SSR in the intergenic region. cerasiforme accession PI 114490 is caused by loss-of-function of SlPSY1 due to trans-splicing between SlPsy1 and an unknown gene transcribed from neighbour opposite strand DNA of SlPsy1. Previous study finds that the yellow fruit in Solanum lycopersicum var. Phytoene synthase (PSY) has been considered as an important regulatory enzyme in carotenoids biosynthesis pathway.
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