Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8–15 nucleotides and by the incorporation of high affinity RNA binders such as 2′, 4′-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2′,4′-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4–5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.     

The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function

Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES–40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES–RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA–protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES–RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S–3′ UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES–RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.     

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA1c sample

Glycated hemoglobin (HbA_1c) is a ‘gold standard’ biomarker for assessing the glycemic index of an individual. HbA_1c is formed due to nonenzymatic glycosylation at N-terminal valine residue of the β-globin chain. Cation exchange based high performance liquid chromatography (CE–HPLC) is mostly used to quantify HbA_1c in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE–HPLC-based quantification, resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA_1c (52.1%) in the CE–HPLC method. The observed HbA_1c did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA_1c and Hb Beckman might have resulted in the coelution of the variant with HbA_1c in CE–HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA_1c, it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA_1c must be estimated using an alternative method.     

Sequencing of 15 622 gene‐bearing BACs clarifies the gene‐dense regions of the barley genome

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.     

New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii

Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to form oxalacetate. It is composed of three subunits, the central cylindrical hexameric 12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here report the cloning, sequencing, expression, and purification of the 5S subunit. The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database. For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a protein of 505 amino acids and of M(r) 55,700.     

Analysis of spatial distribution of genetic diversity and validation of Indian foxtail millet core collection

Foxtail millet [Setaria italica (L.) P. Beauv.] is an important small millet, grown as a short duration, drought tolerant crop across the world. This crop can be grown on wide ranges of soil conditions and has an immense potential for food and fodder in rainfed and arid regions of the India. In the present study, 31 primer pairs (27 SSR and 4 EST-SSR) were used to analyse the genetic diversity in 223 core collection accessions. Analysis resulted in detection of a total of 136 alleles with an average of 4.38 alleles per locus. Among these 136 alleles, 22 were rare, 70 were common and 44 were frequent. The PIC value ranged from 0.01 to 0.86 with an average of 0.31. The average number of observed alleles ranged from 2.0 (northern hills of India accessions) to 4.06 (exotic) with an average of 2.72. The mean Shannon’s Information Index ranged from 0.44 (northern hills of India) to 0.69 (exotic) with an average of 0.52. Pair-wise Fst values indicated little to moderate genetic differentiation among the group of accessions. UPGMA clustering grouped the accessions into two major groups while analysis for population substructure indicated presence of four subpopulations. However there was no statistically well supported grouping of the accessions based on eco-geographic specificities. The core collection designated here represented substantial genetic diversity at molecular level, hence may be a good source of diversity for use in foxtail improvement programs in the region.     

Allozymic variation in four Indian species of genusMus: A comparative analysis

The present study focusses on allozyme variation in the commensal house mouseMus musculus, the pygmy field miceM. booduga andM. terricolor, and the spiny mouseM. platythrix. Genetic heterozygosity was estimated using a set of 24 polymorphic biochemical genetic markers. The extent of variability present inM. booduga, M. terricolor andM. platythrix has been compared with that in theM. musculus complex. Levels of allozyme variation at species level indicate thatM. musculus has the maximum heterogeneity, followed byM. booduga andM. terricolor, whileM. platythrix shows comparatively homogeneous genetic make-up. Gene frequency data have been used to trace phylogenetic relationships among these four species.     

Standardization of in vitro micropropagation procedure of Oriental Lilium Hybrid Cv. ‘Ravenna’

Micropropagation protocol of Oriental Hybrid Lilium cv. Ravenna was developed using bulb scale segments (Basal and Tip) as explants. Surface sterilization of healthy bulb scales with carbendazim 200 ppm for 30 min, then 0.1 percent mercuric chloride for 10 min, then 70% ethyl alcohol for 30 s was superior to all other treatments in recording highest culture asepsis (77.08%) and higher explant survival (86.12%). Explant survival was higher in basal segments (88.54%) compared to tip segments (85.52%). Highest culture establishment was recorded in basal scale segments (68.26%) followed by tip scale segments (55.21%). MS medium augmented with 0.50 mgl⁻¹ Naphthalene acetic acid and 2.0 mgl⁻¹. 6-Benzylamino Purine recorded maximum culture establishment (76.17%), highest bulblet number/explant (5.52) with maximum length of shoots (2.20 cm) and number of leaves (3.39). This treatment combination of growth regulators resulted in highest shoot proliferation (83.33%) along with maximum shoot number (2.41explant⁻¹), shoot length (2.35 cm) and leaf number (5.44) of micro shoots during proliferation stage. Rooting of explants was superior with Indole-3-butyric acid compared to Naphthalene acetic acid. Highest rooting of 92.71% along with maximum number of primary roots shoot⁻¹ (12.06), maximum primary root length (3.17 cm) was documented in Murashige and Skoog medium added with Indole-3-butyric acid 1.50 mgl⁻¹ with best ex vitro survival rate (98.96%) of rooted plantlets during primary hardening in perlite + vermiculite (1:1) mixture.