Postsynthetic On-Column 2′ Functionalization of RNA by Convenient Versatile Method

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2′ position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2′-O-protecting groups, namely 2′-O-thiomorpholine-carbothioate (TC, as “permanent”) and 2′-O-tert-butyl(dimethyl)silyl (tBDMS, as “temporary”), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2′ functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2′ functionalities were synthesized and studied with respect to their physicochemical properties

Supplementary material from Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique

Supplementary figures for all experiments presented in “Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique

Method for the Isolation of “RNA-seq-Quality” RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization

The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells

The Mutation Spectrum of Maturity Onset Diabetes of the Young (MODY)-Associated Genes among Western Siberia Patients

Maturity onset diabetes of the young (MODY) is a congenital form of diabetes characterized by onset at a young age and a primary defect in pancreatic-β-cell function. Currently, 14 subtypes of MODY are known, and each is associated with mutations in a specific gene: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11, ABCC8, and APPL1. The most common subtypes of MODY are associated with mutations in the genes GCK, HNF1A, HNF4A, and HNF1B. Among them, up to 70% of cases are caused by mutations in GCK and HNF1A. Here, an analysis of 14 MODY genes was performed in 178 patients with a MODY phenotype in Western Siberia. Multiplex ligation-dependent probe amplification analysis of DNA samples from 50 randomly selected patients without detectable mutations did not reveal large rearrangements in the MODY genes. In 38 patients (37% males) among the 178 subjects, mutations were identified in HNF4A, GCK, HNF1A, and ABCC8. We identified novel potentially causative mutations p.Lys142*, Leu146Val, Ala173Glnfs*30, Val181Asp, Gly261Ala, IVS7 c.864 −1G>T, Cys371*, and Glu443Lys in GCK and Ser6Arg, IVS 2 c.526 +1 G>T, IVS3 c.713 +2 T>A, and Arg238Lys in HNF1A