Targeting of CRISPR-Cas12a crRNAs into human mitochondria.

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3'-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.

Silver Nanoparticles Modified by Carbosilane Dendrons and PEG as Delivery Vectors of Small Interfering RNA.

The fact that cancer is one of the leading causes of death requires researchers to create new systems of effective treatment for malignant tumors. One promising area is genetic therapy that uses small interfering RNA (siRNA). These molecules are capable of blocking mutant proteins in cells, but require specific systems that will deliver RNA to target cells and successfully release them into the cytoplasm. Dendronized and PEGylated silver nanoparticles as potential vectors for proapoptotic siRNA (siMCL-1) were used here. Using the methods of one-dimensional gel electrophoresis, the zeta potential, dynamic light scattering, and circular dichroism, stable siRNA and AgNP complexes were obtained. Data gathered using multicolor flow cytometry showed that AgNPs are able to deliver (up to 90%) siRNAs efficiently to some types of tumor cells, depending on the degree of PEGylation. Analysis of cell death showed that complexes of some AgNP variations with siMCL-1 lead to ~70% cell death in the populations that uptake these complexes due to apoptosis.

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support.

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.

Simplistic perylene-related compounds as inhibitors of tick-borne encephalitis virus reproduction.

Rigid amphipathic fusion inhibitors are potent broad-spectrum antivirals based on the perylene scaffold, usually decorated with a hydrophilic group linked via ethynyl or triazole. We have sequentially simplified these structures by removing sugar moiety, then converting uridine to aniline, then moving to perylenylthiophenecarboxylic acids and to perylenylcarboxylic acid. All these polyaromatic compounds, as well as antibiotic heliomycin, still showed pronounced activity against tick-borne encephalitis virus (TBEV) with limited toxicity in porcine embryo kidney (PEK) cell line. 5-(Perylen-3-yl)-2-thiophenecarboxylic acid (5a) showed the highest antiviral activity with 50% effective concentration of approx. 1.6 nM.

Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo.

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.

Antivirals acting on viral envelopes via biophysical mechanisms of action.

Most antivirals target viral proteins and are specific for only one virus, or viral type. Whereas viral proteins are encoded in the plastic viral genome, virion lipids are not and their rearrangements during fusion are conserved among otherwise unrelated enveloped viruses. Antivirals that inhibit these lipid rearrangements could thus pose a high barrier to resistance and have broad-spectrum activity. Fusion occurs through a hemifusion stalk in which only the outer leaflets are fused and thus curved with a smaller radius for the polar heads than for the hydrophobic tails (negative curvature). Outer leaflets enriched in phospholipids with head groups of larger cross sections than their lipid tails ("inverted cone") disfavor negative curvature, inhibiting fusion. The rigid amphipathic fusion inhibitors (RAFIs) are synthetic compounds of inverted cone molecular geometry. They inhibit infectivity of otherwise unrelated enveloped viruses. The leading RAFI, aUY11, has an ethynyl-perylene hydrophobic and an uracil-arabinose polar moiety. aUY11 intercalates in viral envelopes and inhibits virion-to-cell fusion of a broad spectrum of otherwise unrelated enveloped viruses. Previous studies showed that amphipathicity, rigidity, and inverted cone molecular geometry were required. We propose that the inverted cone molecular geometry of the RAFIs increases the energy barrier for the hemifusion stalk, inhibiting fusion. Then, chemically distinct compounds with similar amphipathicity, rigidity, and inverted cone shape would have similar antiviral potencies, regardless of specific chemical groups. Alternatively, the perylene group exposed to visible light may induce viral lipid peroxidation. Then, the perylene group and absorbance at visible spectrum would be required. We now evaluated twenty-five chemically distinct RAFIs. The perylene moiety and absorption at visible spectrum were not required, but a minimum length of the hydrophobic moiety was, 10.3 Å. The arabino moiety could be modified or replaced by other groups. Cytidine was not tolerated. Bilayer intercalation was required but not sufficient. The vast majority of RAFIs had no overt cytotoxicity (CC50 > 20 µM; TI > 250-1200). Carbonyl or butylamide substitutions for arabino, or cytidine replacement for uracil, increased cytotoxicity. Cytotoxicity was mainly determined by the polar moiety and there was no correlation between antiviral and cytostatic activities. The definition of the effects of shape and chemical groups of the RAFIs opens the possibility to the rational design of lipid-acting antivirals active against a broad spectrum of enveloped viruses.

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

CyTOF Mass Cytometry for Click Proliferation Assays.

Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S-phase determination in cell cycle applications, combining well-established click chemistry with a novel iodo-deoxyuridine (IdU) azide derivative and a cross-reactive anti-IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU-based assays for complex and multiparametric experiments. It provides a feasible cost-effective approach for detecting ethynyl-labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine.

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 ß-elimination contributes to the rate-limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59-1.83 fold change in the rate of catalysis, thus suggesting that ß-elimination is partly rate-limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing ß-elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.