Nucleic Acids Research最新文献

Oligonucleotide (ON) therapeutics are promising as disease-modifying therapies for central nervous system (CNS) disorders. Intrathecal ON administration into the cerebral spinal fluid is a safe and effective delivery mode to the CNS. However, preclinical studies have shown acute and transient changes in neurobehavior following high-dose central ON delivery. Here, we characterize a subset of these changes peaking 15 min after ON dosing and resolving after 120 min. Symptoms include shaking, muscle twitching, cramping, hyperactivity, hyperreactivity, vocalizations, tremors, convulsions, and seizures. These are collectively referred to as the acute neuronal activation response. Acute neuronal activation is observed in rats, mice, and nonhuman primates and is quantifiable using a simple scoring system. It is distinct from acute inhibition seen with some phosphorothioate-modified antisense oligonucleotides, characterized by loss of spinal reflexes, ataxia, and sedation. The acute neuronal activation response is largely sequence-independent and is driven by ON chelation of divalent cations, particularly influenced by the divalent cation-to-ON ratio in the dosing solution. Acute neuronal activation can be safely mitigated by adjusting this ratio through magnesium supplementation in the ON formulation. We provide a comprehensive framework for quantifying and mitigating the acute neuronal activation response caused by high concentrations of centrally delivered ON therapeutics in preclinical species.

Some naturally occurring ribozymes can catalyze self-cleavage reactions through a 2'-OH group. Consequently, experimental structures of pre-catalytic states often require chemical modifications of the 2'-OH, such as its removal or methylation. However, the impact of these chemical modifications on the active site structure remains largely unexplored, which raises important questions since methylated structures are often taken as being representative of pre-catalytic states. Here, we employ extensive atomistic simulations critically compared with and fine-tuned on experimental data, and we revisit experimental results to show that 2'-O-methylation critically affects reactant geometries and, therefore, the possible reaction mechanisms inferred from the structures. Our results also challenge the common assumption that 2'-O-methylation stabilizes the C3'-endo puckering conformation. Our findings, consistent with recent experimental data on ribosome structure, reveal that this effect is highly sensitive to the local secondary structure and is often overstated. For three investigated small-cleaving ribozymes, the C2'-endo conformation observed for chemically modified active site residues through 2'-O-methylation is not stable upon methyl group removal to obtain the catalytically relevant hydroxylated state. This suggests that these geometries arise primarily from a combination of steric hindrances and electrostatic interactions with the surrounding environment rather than intrinsic conformational preferences of the ribose upon methylation.

The impacts of various stressors on bacterial systems have been studied at the phenotypic, transcriptional, and translational levels during the early stress response. However, the contributions of RNA modifications during stress adaptation remain largely unexplored. Here, we map the epitranscriptomic changes of Escherichia coli after exposure to oxidative and acid stress using direct RNA sequencing of mRNA, rRNA, pre-tRNA, and tRNA, combined with mass spectrometry, deletion mutant phenotyping, and single-nucleotide PCR. We identified widespread, dynamic mRNA modifications that include central metabolism transcripts and increased levels of rRNA methylations (m4Cm and m5C) under both stresses, with potential consequences for translation. In uncharged pre-tRNAs, stress-specific modifications via the Mnm and Q pathways accumulated at the wobble position; these modifications proved crucial for survival. Together, these findings reveal a multifaceted layer of post-transcriptional regulation, establishing the first comprehensive view of the bacterial epitranscriptome during the early stress response.

Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, the role of enhancer looping protein LIM domain-binding 1 (LDB1) in stem cells remains to be fully explored. We generated Ldb1(-/-) embryonic stem cells (ESCs) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Differential gene expression, including of the Lin28-mediated self-renewal pathway genes, was observed between wild-type and Ldb1(-/-) ESC. LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors, and LDB1 loss resulted in loss of Sox2 interactions with the SCR enhancer. Embryoid bodies (EBs) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers. Ldb1(-/-) ESC had impaired ability to undergo terminal differentiation to erythroblasts, and gene dysregulation was very pronounced in Ldb1(-/-) erythroblasts. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important for normal gene expression during differentiation to erythroblasts.