Proximity-Induced Nucleic Acid Degrader (PINAD) Approach to Targeted RNA Degradation Using Small Molecules.

Nature has evolved intricate machinery to target and degrade RNA, and some of these molecular mechanisms can be adapted for therapeutic use. Small interfering RNAs and RNase H-inducing oligonucleotides have yielded therapeutic agents against diseases that cannot be tackled using protein-centered approaches. Because these therapeutic agents are nucleic acid-based, they have several inherent drawbacks which include poor cellular uptake and stability. Here we report a new approach to target and degrade RNA using small molecules, proximity-induced nucleic acid degrader (PINAD). We have utilized this strategy to design two families of RNA degraders which target two different RNA structures within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral pseudoknot. We demonstrate that these novel molecules degrade their targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy allows any RNA binding small molecule to be converted into a degrader, empowering RNA binders that are not potent enough to exert a phenotypic effect on their own. PINAD raises the possibility of targeting and destroying any disease-related RNA species, which can greatly expand the space of druggable targets and diseases.

Examining the Potential Synergistic Effects Between Mindfulness Training and Psychedelic-Assisted Therapy.

Mindfulness-based interventions and psychedelic-assisted therapy have been experimentally utilised in recent years as alternative treatments for various psychopathologies with moderate to great success. Both have also demonstrated significant post-acute and long-term decreases in clinical symptoms and enhancements in well-being in healthy participants. These two therapeutic interventions share various postulated salutogenic mechanisms, such as the ability to alter present-moment awareness and anti-depressive action, via corresponding neuromodulatory effects. Recent preliminary evidence has also demonstrated that psychedelic administration can enhance mindfulness capacities which has already been demonstrated robustly as a result of mindfulness-based interventions. These shared mechanisms between mindfulness-based interventions and psychedelic therapy have led to scientists theorising, and recently demonstrating, synergistic effects when both are used in combination, in the form of potentiated therapeutic benefit. These synergistic results hold great promise but require replication in bigger sample groups and better controlled methodologies, to fully delineate the effect of set and setting, before they can be extended onto clinical populations.

Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

Modified Forms of Cytosine in Eukaryotes: DNA (De)methylation and Beyond.

5-Methylcytosine (5mC) is an epigenetic mark known to contribute to the regulation of gene expression in a wide range of biological systems. Ten Eleven Translocation (TET) dioxygenases oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in metazoans and fungi. Moreover, two recent reports imply the existence of other species of modified cytosine in unicellular alga Chlamydomonas reinhardtii and malaria parasite Plasmodium falciparum. Here we provide an overview of the spectrum of cytosine modifications and their roles in demethylation of DNA and regulation of gene expression in different eukaryotic organisms.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

5-Carboxylcytosine levels are elevated in human breast cancers and gliomas.

BACKGROUND: DNA methylation (5-methylcytosine (5mC)) patterns are often altered in cancers. Ten-eleven translocation (Tet) proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). In addition to their presumptive specific biological roles, these oxidised forms of 5mC may serve as intermediates in demethylation process. According to several reports, 5hmC levels are strongly decreased in cancers; however, the distribution of 5fC and 5caC in malignant tissue has not been studied. FINDINGS: Here, we examine the levels of 5hmC and 5caC in 28 samples of normal breast tissue, 59 samples of invasive human breast cancer and 74 samples of gliomas using immunochemistry. In agreement with previous reports, we show that 71 % of normal breast samples exhibit strong 5hmC signal, compared with only 18 % of breast cancer samples with equivalent levels of 5hmC staining. Unexpectedly, although 5caC is not detectable in normal breast tissue, 27 % of breast cancer samples exhibit significant staining for this modification (p < 0.001). Surprisingly, the presence of immunochemically detectable 5caC is not associated with the intensity of 5hmC signal in breast cancer tissue. In gliomas, we show that 5caC is detectable in 45 % of tumours. CONCLUSIONS: We demonstrate that, unlike 5hmC, the levels of 5caC are elevated in a proportion of breast cancers and gliomas. Our results reveal another level of complexity to the cancer epigenome, suggesting that active demethylation and/or 5caC-dependent transcriptional regulation are pre-activated in some tumours and may contribute to their pathogenesis. Larger studies to evaluate the clinicopathological significance of 5caC in cancers are warranted.

Surface magnetoinductive breathers in two-dimensional magnetic metamaterials.

We study discrete surface breathers in two-dimensional lattices of inductively coupled split-ring resonators with capacitive nonlinearity. We consider both conservative (Hamiltonian) and analyze the properties of the modes localized in space and periodic in time (discrete breathers) located in the corners and on the edges of the lattice. We find that surface breathers in the Hamiltonian systems have lower energy than their bulk counterparts and they are generally more stable.

Destruction of long-range interactions by a single mutation in lysozyme.

We propose a mechanism, based on a > or =10-micros molecular dynamics simulation, for the surprising misfolding of hen egg-white lysozyme caused by a single mutation (W62G). Our simulations of the wild-type and mutant lysozymes in 8 M urea solution at biological temperature (with both pH 2 and 7) reveal that the mutant structure is much less stable than that of the wild type, with the mutant showing larger fluctuations and less native-like contacts. Analysis of local contacts reveals that the Trp-62 residue is the key to a cooperative long-range interaction within the wild type, where it acts like a bridge between two neighboring basic residues. Thus, a native-like cluster or nucleation site can form near these residues in the wild type but not in the mutant. The time evolution of the secondary structure also exhibits a quicker loss of the beta-sheets in the mutant than in the wild type, whereas some of the alpha-helices persist during the entire simulation in both the wild type and the mutant in 8 M urea (even though the tertiary structures are basically all gone). These findings, while supporting the general conclusions of a recent experimental study by Dobson and coworkers [Klein-Seetharam J, Oikama M, Grimshaw SB, Wirmer J, Duchardt E, Ueda T, Imoto T, Smith LJ, Dobson CM, Schwalbe H (2002) Science 295:1719-1722], provide a detailed but different molecular picture of the misfolding mechanism.

Thermal denaturing of mutant lysozyme with both the OPLSAA and the CHARMM force fields.

Biomolecular simulations enabled by massively parallel supercomputers such as BlueGene/L promise to bridge the gap between the currently accessible simulation time scale and the experimental time scale for many important protein folding processes. In this study, molecular dynamics simulations were carried out for both the wild-type and the mutant hen lysozyme (TRP62GLY) to study the single mutation effect on lysozyme stability and misfolding. Our thermal denaturing simulations at 400-500 K with both the OPLSAA and the CHARMM force fields show that the mutant structure is indeed much less stable than the wild-type, which is consistent with the recent urea denaturing experiment (Dobson et al. Science 2002, 295, 1719-1722; Nature 2003, 424, 783-788). Detailed results also reveal that the single mutation TRP62GLY first induces the loss of native contacts in the beta-domain region of the lysozyme protein at high temperatures, and then the unfolding process spreads into the alpha-domain region through Helix C. Even though the OPLSAA force field in general shows a more stable protein structure than does the CHARMM force field at high temperatures, the two force fields examined here display qualitatively similar results for the misfolding process, indicating that the thermal denaturing of the single mutation is robust and reproducible with various modern force fields.

Hydration and dewetting near fluorinated superhydrophobic plates.

The water dynamics near nanoscale fluorinated (CF(3)(CF(2))(7)(CH(2))(2)SiH(3)) monolayers (plates) as well as possible dewetting transitions in-between two such plates have been studied with molecular dynamics simulations in this paper. A "weak water depletion" is found near the single fluorinated surface, with an average water density in the first solvation shells 6-8% lower than its hydrogenated counterpart. The fluorinated molecules are also found to be water impermeable, consistent with experimental findings. More surprisingly, a dewetting transition is found in the interplate region with a critical distance D(c) of 10 A (3-4 water diameters) for double plates with 8 x 8 molecules each (plate size approximately 4 nm x 4 nm). This transition, although occurring on a microscopic length scale, is reminiscent of a first-order phase transition from liquid to vapor. The unusual superhydrophobicity of fluorocarbons is found to be related to their larger size (or surface area) as compared to hydrocarbons, which "dilutes" their physical interactions with water. The water-plate interaction profile shows that the fluorinated carbons have a 10-12% weaker water-plate interaction than their hydrogenated counterparts in the nearest solvation shell, even though the fluorocarbons do have a stronger electrostatic interaction with water due to their larger partial charges. However, the van der Waals interactions dominate the water-plate interaction within the nearest shell, with up to 90% contributions to the total interaction energy, and fluorocarbons have a noticeably weaker (by 10-15%) van der Waals interaction with water in the nearest shell than do hydrocarbons. Both the slightly weaker water-plate interaction and larger surface area contribute to the stronger dewetting transition in the current fluorinated carbon plates.