Polymerase-directed synthesis of C5-ethynyl locked nucleic acids

Modified nucleic acids have considerable potential in nanobiotechnology for the development of nanomedicines and new materials. Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far and we herein for the first time report the enzymatic incorporation of LNA-U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5′-triphosphate as substrate for RNA transcripts

BACE1 inhibition using 2’-OMePS steric blocking antisense oligonucleotides

Amyloid beta-peptide is produced by the cleavage of amyloid precursor protein by two secretases, a β-secretase, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and a γ-secretase. It has been hypothesised that partial inhibition of BACE1 in individuals with a high risk of developing Alzheimer’s disease may be beneficial in preventing cognitive decline. In this study, we report the development of a novel antisense oligonucleotide (AO) that could efficiently downregulate the BACE1 transcript and partially inhibit BACE1 protein. We designed and synthesised a range of 2’-OMethyl-modified antisense oligonucleotides with a phosphorothioate backbone across various exons of the BACE1 transcript, of which AO2, targeting exon 2, efficiently downregulated BACE1 RNA expression by 90%. The sequence of AO2 was later synthesised with a phosphorodiamidate morpholino chemistry, which was found to be not as efficient at downregulating BACE1 expression as the 2’-OMethyl antisense oligonucleotides with a phosphorothioate backbone variant. AO2 also reduced BACE1 protein levels by 45%. In line with our results, we firmly believe that AO2 could be used as a potential preventative therapeutic strategy for Alzheimer’s diseas

Genetic analysis of age at conception as a fertility proxy in ewe lambs of Norwegian White Sheep: heritability and genetic correlation with body growth and leanness

The aim of this study was to estimate the genetic variation and heritability of ewe’s age at first lambing (a fertility proxy) in ewe lambs of Norwegian White Sheep and to estimate the genetic correlations between number of days till lambing and, respectively, growth rate and fat deposition at slaughter (EUROP classification score) in half year-old ewes, utilizing data on the ewe itself or on sibs (for fat deposition). The data of this study is from the breed Norwegian white sheep (NWS) and was made available by Animalia. From the original dataset one dataset was sampled containing 91683 females with weaning weight information and amongst these 31002 ewes with information for number of days at lambing. Pedigree files traced back to third generation were used in the analysis. Age at litter, the fertility success trait showed considerable heritability of 0.08 and had a genetic and environmental correlation of zero with the most focused traits in the Norwegian sheep breeding program; growth rate and fat score. Therefore, we conclude that farmers can select this fertility success trait without affecting the current objectives of the breeding goal. On the contrary, such a strategy would make positive steps towards economic and environmental sustainability.The aim of this study was to estimate the genetic variation and heritability of ewe’s age at first lambing (a fertility proxy) in ewe lambs of Norwegian White Sheep and to estimate the genetic correlations between number of days till lambing and, respectively, growth rate and fat deposition at slaughter (EUROP classification score) in half year-old ewes, utilizing data on the ewe itself or on sibs (for fat deposition). The data of this study is from the breed Norwegian white sheep (NWS) and was made available by Animalia. From the original dataset one dataset was sampled containing 91683 females with weaning weight information and amongst these 31002 ewes with information for number of days at lambing. Pedigree files traced back to third generation were used in the analysis. Age at litter, the fertility success trait showed considerable heritability of 0.08 and had a genetic and environmental correlation of zero with the most focused traits in the Norwegian sheep breeding program; growth rate and fat score. Therefore, we conclude that farmers can select this fertility success trait without affecting the current objectives of the breeding goal. On the contrary, such a strategy would make positive steps towards economic and environmental sustainability

Locked nucleic acid oligonucleotides towards clinical applications

Nucleic acid–based therapeutic technologies (Figure 9.1) have significantly advanced in the past two decades toward the treatment of many diseases. The first such drug to enter clinic was vitravene®, an antisense oligonucleotide for the treatment of cytomegalovirus retinitis [1]. Later, research on aptamers led to the marketing of macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD) [2]. Nucleic acid–based therapeutic approaches mainly include antisense [3,4], ribozymes [4], small interfering RNA (siRNA) [4–6], microRNA (miRNA) [7–10] targeting and aptamers [11–15]. Oligonucleotides composed of naturally occurring DNA or RNA nucleotides pose some limitations because of their poor RNA binding affinity, low degree of nuclease resistance, and low bioavilability. To overcome these limitations, chemically modified nucleic acids have been introduced, among which locked nucleic acid (LNA) [16–20] proved to be unique and is now used extensively for various applications in chemical biology [21–23]

Locked nucleic acid as a novel class of therapeutic agents

Locked Nucleic Acid (LNA) is a nucleic acid analogue with unprecedented binding affinity and excellent specificity toward complementary RNA and DNA oligonucleotides. The remarkable properties of LNA have led to applications within various gene silencing strategies both in vitro and in vivo. In the present review, we highlight the uses of LNA for regulation of gene expression with emphasis on RNA targeting

Next-Generation Nucleic Acid Aptamers with Two-Base-Modified Nucleotides Have Improved Binding Affinity and Potency

Twice as apt: Nucleic acid aptamers with high binding affinity, specificity, epitope coverage and nuclease resistance were developed by using libraries containing oligonucleotides in which two bases in the pyrimidine nucleotide had been modified

Topology Identification under Spatially Correlated Noise

This article addresses the problem of reconstructing the topology of a network of agents interacting via linear dynamics, while being excited by exogenous stochastic sources that are possibly correlated across the agents, from time-series measurements alone. It is shown, under the assumption that the correlations are affine in nature, such network of nodal interactions is equivalent to a network with added agents, represented by nodes that are latent, where no corresponding time-series measurements are available; however, here all exogenous excitements are spatially (that is, across agents) uncorrelated. Generalizing affine correlations, it is shown that, under polynomial correlations, the latent nodes in the expanded networks can be excited by clusters of noise sources, where the clusters are uncorrelated with each other. The clusters can be replaced with a single noise source if the latent nodes are allowed to have non-linear interactions. Finally, using the sparse plus low-rank matrix decomposition of the imaginary part of the inverse power spectral density matrix (IPSDM) of the time-series data, the topology of the network is reconstructed. Under non conservative assumptions, the correlation graph is retrieved.Comment: 14 pages, 5 figure

Development of novel antimiRzymes for targeted inhibition of miR-21 expression in solid cancer cells

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the regulation of gene expression. Previous reports showed an over-expression of miRNA-21 (miR-21) in various cancer cells, and its up-regulation is closely related to cancer initiation, proliferation and metastasis. In this work, we envisioned the development of novel antimiRzymes (anti-miRNA-DNAzyme) that are capable of selectively targeting and cleaving miR-21 and inhibit its expression in cancer cells using the DNAzyme technique. For this purpose, we have designed different antimiRzyme candidates by systematically targeting different regions of miR-21. Our results demonstrated that RNV541, a potential arm-loop-arm type antimiRzyme, was very efficient (90%) to suppress miR-21 expression in U87MG malignant glioblastoma cell line at 200 nM concentration. In addition, RNV541 also inhibited miR-21 expression (50%) in MDA-MB-231 breast cancer cell line. For targeted delivery, we conjugated RNV541 with a transferrin receptor (TfR) targeting aptamer for TfR-mediated cancer cell delivery. As expected, the developed chimeric structure efficiently delivered the antimiRzyme RNV541 into TfR positive glioblastoma cells. TfR aptamer-RNV541 chimeric construct showed 52% inhibition of miR-21 expression in U87MG glioblastoma cells at 2000 nM concentration, without using any transfection reagents, making it a highly desirable strategy to tackle miR-21 over-expressed malignant cancers. Although these are in vitro based observations, based on our results, we firmly believe that our findings could be beneficial towards the development of targeted cancer therapeutics where conventional therapies face several challenges

Novel disulfide-bridged bioresponsive antisense oligonucleotide induces efficient splice modulation in muscle Myotubes in Vitro

Splice-modulating antisense therapy has shown tremendous potential in therapeutic development in recent years with four FDA-approved antisense drugs since 2016. However, an efficient and nontoxic antisense oligonucleotide (AO) delivery system still remains as a major obstacle in nucleic acid therapeutics field. Vitamin-E (α-tocopherol) is an essential dietary requirement for human body. This fat-soluble compound is one of the most important antioxidants which involves in numerous biological pathways. In this study, for the first time, we explored the scope of using α-tocopherol-conjugated bioresponsive AOs to induce splice modulation in mouse muscle myotubes in vitro. Our results showed that the bioresponsive construct efficiently internalized into the cell nucleus and induced exon 23 skipping in mdx mouse myotubes. Based on our exciting new results, we firmly believe that our findings could potentially benefit toward establishing a delivery approach to advance the field of splice-modulating AO therapy