Mutations in microRNA-128-2-3p identified with amplification-free hybridization assay.

We describe a quantitative detection method for mutated microRNA in human plasma samples. Specific oligonucleotides designed from a Peyrard-Bishop model allowed accurate prediction of target:probe recognition affinity and specificity. Our amplification-free tandem bead-based hybridization assay had limit of detection of 2.2 pM. Thereby, the assay allowed identification of single-nucleotide polymorphism mismatch profiles in clinically relevant microRNA-128-2-3p, showing terminal mutations that correlate positively with inflammatory colitis and colorectal cancer.

MicroRNA Pools Synthesized Using Tandem Solid-Phase Oligonucleotide Synthesis.

Herein, we describe a new approach to make pools of microRNA targeting breast cancer cells. The microRNA pools were synthesized at once on the same solid support using the "Tandem Oligonucleotide Synthesis" strategy. We make up to four consecutive microRNAs (miR129-1-5p, miR31, miR206, and miR27b-3p) using 2'/3'OAc nucleotide phosphoramidites, with the total length of the pool reaching 88 nucleotides. The developed phosphoramidites, when combined, give a cleavable moiety that separates the microRNAs and is cleaved using standard post-RNA synthesis cleavage conditions. Furthermore, we investigate making branched pools (microRNA dendrimers) versus linear pools as a strategy to further improve the product yields. Our approach provides with microRNA pools in high yields, which is of relevance to the growing demand on synthetic RNA oligomers for nucleic acid research and technology.

Cation valence dependence of hydrogen bond and stacking potentials in DNA mesoscopic models.

Monovalent and divalent cations play a crucial role in living cells and for molecular techniques such as PCR. Here we evaluate DNA melting temperatures in magnesium (Mg2+) and magnesium­potassium (Mg2++ K+) buffers with a mesoscopic model that allows us to estimate hydrogen bonds and stacking interaction potentials. The Mg2+ and Mg2++ K+ results are compared to previous calculations for sodium ions (Na+), in terms of equivalent sodium concentration and ionic strength. Morse potentials, related to hydrogen bonding, were found to be essentially constant and unaffected by cation conditions. However, for stacking interactions we find a clear dependence with ionic strength and cation valence. The highest ionic strength variations, for both hydrogen bonds and stacking interactions, was found at the sequence terminals. This suggests that end-to-end interactions in DNA will be strongly dependent on cation valence and ionic strength.

New approaches to moderate CRISPR-Cas9 activity: Addressing issues of cellular uptake and endosomal escape.

CRISPR-Cas9 is rapidly entering molecular biology and biomedicine as a promising gene-editing tool. A unique feature of CRISPR-Cas9 is a single-guide RNA directing a Cas9 nuclease toward its genomic target. Herein, we highlight new approaches for improving cellular uptake and endosomal escape of CRISPR-Cas9. As opposed to other recently published works, this review is focused on non-viral carriers as a means to facilitate the cellular uptake of CRISPR-Cas9 through endocytosis. The majority of non-viral carriers, such as gold nanoparticles, polymer nanoparticles, lipid nanoparticles, and nanoscale zeolitic imidazole frameworks, is developed with a focus toward optimizing the endosomal escape of CRISPR-Cas9 by taking advantage of the acidic environment in the late endosomes. Among the most broadly used methods for in vitro and ex vivo ribonucleotide protein transfection are electroporation and microinjection. Thus, other delivery formats are warranted for in vivo delivery of CRISPR-Cas9. Herein, we specifically revise the use of peptide and nanoparticle-based systems as platforms for CRISPR-Cas9 delivery in vivo. Finally, we highlight future perspectives of the CRISPR-Cas9 gene-editing tool and the prospects of using non-viral vectors to improve its bioavailability and therapeutic potential.

Complete Mesoscopic Parameterization of Single LNA Modifications in DNA Applied to Oncogene Probe Design.

The use of mesoscopic models to describe the thermodynamic properties of locked nucleic acid (LNA)-modified nucleotides can provide useful insights into their properties, such as hydrogen-bonding and stacking interactions. In addition, the mesoscopic parameters can be used to optimize LNA insertion in probes, to achieve accurate melting temperature predictions, and to obtain duplex opening profiles at the base-pair level. Here, we applied this type of model to parameterize a large set of melting temperatures for LNA-modified sequences, from published sources, covering all possible nearest-neighbor configurations. We have found a very large increase in Morse potentials, which indicates very strong hydrogen bonding as the main cause of improved LNA thermodynamic stability. LNA-modified adenine-thymine (AT) was found to have similar hydrogen bonding to unmodified cytosine-guanine (CG) base pairs, while for LNA CG, we found exceptionally large hydrogen bonding. In contrast, stacking interactions, which were thought to be behind the stability of LNA, were similar to unmodified DNA in most cases. We applied the new LNA parameters to the design of BRAF, KRAS, and EGFR oncogene variants by testing all possible LNA modifications. Selected sequences were then synthesized and had their hybridization temperatures measured, achieving a prediction accuracy within 1 °C. We performed a detailed base-pair opening analysis to discuss specific aspects of these probe hybridizations that may be relevant for probe design.

Carbon Nanotubes-Potent Carriers for Targeted Drug Delivery in Rheumatoid Arthritis.

Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77-79% for HiPco-SWCNT and 71-83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90-97% for HiPco-SWCNT and 87-98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000-1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

Control of LDL Uptake in Human Cells by Targeting the LDLR Regulatory Long Non-coding RNA BM450697.

Hypercholesterolemia is a condition that is characterized by very high levels of cholesterol in the blood and is a major correlating factor with heart disease. Indeed, high levels of the low-density lipoprotein (LDL) have been causally linked to the development of atherosclerotic cardiovascular disease (ASCVD). A method to specifically reduce cholesterol in the blood in a long-term, stable manner could prove therapeutically relevant. Cholesterol is removed from the blood by the LDL receptor (LDLR) in the liver. Others and we have discovered that a long non-coding RNA (lncRNA; BM450697) functions as an endogenous epigenetic regulator of LDLR and that the repression of this lncRNA by the action of small interfering RNAs (siRNAs) results in the activation of LDLR. We found here, through the interrogation of two siRNAs that can target this lncRNA, both in a transcriptional and post-transcriptional manner, that BM450697 functions as a local scaffold for modulating LDLR transcription. Moreover, we found that conjugation of α-N-acetylgalactosamine (GalNAc) with two lncRNA-directed siRNAs allows for direct liver cell targeting of this lncRNA and functional enhanced uptake of cholesterol. Collectively, these data suggest that targeting the BM450697 lncRNA regulator of LDLR may result in a more specific, long-term, targeted approach to regulating cholesterol in the blood.

Discovery and description of a new serogroup 7 Streptococcus pneumoniae serotype, 7D, and structural analysis of 7C and 7D.

Streptococcus pneumoniae is characterised into 92 serotypes based on antigenic reactions of commercial rabbit sera to the capsular polysaccharides. During development of a bioinformatic serotyping tool (PneumoCaT), an isolate exhibited a novel codon at residue 385 of the glycosyltransferase gene wcwK encoding a distinct amino acid, which differentiates genogroup 7. Investigation by repeat serotyping and Quellung reaction revealed a novel pattern of factor sera with the isolate reacting very strongly with 7f, but also with 7e factor sera. The structure of the capsular polysaccharide was determined by NMR spectroscopy to be an approximately 5:1 combination of the structures of 7C and 7B, respectively, and the structure of 7C was also elucidated. All data from whole genome sequencing, NMR spectroscopy, production of antisera and serotyping of the novel 7 strain shows that it is a new serotype, which will be named in the Danish nomenclature as 7D.