Systematic Analysis of 2'-O-Alkyl Modified Analogs for Enzymatic Synthesis and Their Oligonucleotide Properties.

Enzymatic oligonucleotide synthesis is used for the development of functional oligonucleotides selected by in vitro selection. Expanding available sugar modifications for in vitro selection helps the functional oligonucleotides to be used as therapeutics reagents. We previously developed a KOD DNA polymerase mutant, KOD DGLNK, that enzymatically synthesized fully-LNA- or 2'-O-methyl-modified oligonucleotides. Here, we report a further expansion of the available 2'-O-alkyl-modified nucleotide for enzymatic synthesis by KOD DGLNK. We chemically synthesized five 2'-O-alkyl-5-methyluridine triphosphates and incorporated them into the oligonucleotides. We also enzymatically synthesized a 2'-O-alkyl-modified oligonucleotide with a random region (oligonucleotide libraries). The 2'-O-alkyl-modified oligonucleotide libraries showed high nuclease resistance and a wide range of hydrophobicity. Our synthesized 2'-O-alkyl-modified oligonucleotide libraries provide novel possibilities that can promote the development of functional molecules for therapeutic use.

Detection of circulating hepatitis B virus immune escape and polymerase mutants among HBV-positive patients attending Institut Pasteur de Bangui, Central African Republic.

BACKGROUND: Previous studies in the Central African Republic (CAR) have reported the presence of hepatitis B virus (HBV) recombinant genotype E/D and a suspicion of immune escape mutants (IEMs), without further investigation into their impact on prevention and diagnosis. Consequently, this study investigated HBV mutations among hepatitis B surface antigen (HBsAg)-positive patients attending Institut Pasteur de Bangui in the CAR. METHODS: Sera from a total of 118 HBsAg-positive patients with no previous history of HBV treatment or vaccination at the Institut Pasteur de Bangui, were sampled between 2017 and 2019. Subsequently, the region spanning the surface and polymerase genes of HBV was amplified by PCR and sequenced. HBV sequences were genotyped/subgenotyped by phylogenetic analysis and serotyped based on predicted amino acid residues at positions s122, s127, s140, s159, and s160. They were then analyzed for HBV IEMs and polymerase mutations. RESULTS: The region spanning the surface and polymerase genes was successfully amplified and sequenced for 51 samples. Of the HBV sequences, 49 were genotype E and two were genotype A subgenotype A1; these were serotyped as ayw4 and ayw1, respectively. Potential IEMs sY100C, sA128V, and sM133T, and several polymerase mutants were identified. CONCLUSIONS: This study raises awareness of the need for further studies to be conducted on a large scale to better understand HBV mutations for improved disease control and prevention strategies in the country.

Targeting flavivirus RNA dependent RNA polymerase through a pyridobenzothiazole inhibitor.

RNA dependent RNA polymerases (RdRp) are essential enzymes for flavivirus replication. Starting from an in silico docking analysis we identified a pyridobenzothiazole compound, HeE1-2Tyr, able to inhibit West Nile and Dengue RdRps activity in vitro, which proved effective against different flaviviruses in cell culture. Crystallographic data show that HeE1-2Tyr binds between the fingers domain and the priming loop of Dengue virus RdRp (Site 1). Conversely, enzyme kinetics, binding studies and mutational analyses suggest that, during the catalytic cycle and assembly of the RdRp-RNA complex, HeE1-2Tyr might be hosted in a distinct binding site (Site 2). RdRp mutational studies, driven by in silico docking analysis, allowed us to locate the inhibition Site 2 in the thumb domain. Taken together, our results provide innovative concepts for optimization of a new class of anti-flavivirus compounds.

Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants.

Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.