These findings identify nucleotide class composition as a previously unrecognized parameter governing IVT-mRNA function and establish hybrid ribonucleotide-deoxyribonucleotide backbone engineering as a versatile strategy to expand the chemical space for next-generation mRNA therapeutics.
Replicative DNA polymerases are responsible for the high-fidelity duplication of genomes in all organisms. The overall DNA replication process is highly accurate, with deoxyribonucleoside monophosphate (dNMP) errors occurring on the order of 1 in tens of millions of replicated bases. However, DNA polymerases are more prone to misincorporating ribonucleoside monophosphates (rNMPs) in place of their cognate dNMPs, with measurements on the order of 1 in a couple thousand base pairs synthesized. Owing in part to the high concentration of rNTPs relative to dNTPs in vivo, rNMPs may represent the most common nucleotide requiring removal from genomic DNA in organisms spanning all domains of life. Cells employ a set of pathways, including ribonucleotide excision repair (RER), to address abundant genomic rNMPs. Defects in ribonucleotide excision repair result in mutagenesis, neurological disorders, and embryonic lethality. In this review, we discuss the mechanisms for how ribonucleotides are misincorporated by DNA polymerases, how RNase H enzymes recognize and cleave RNA-DNA hybrids, and the process of ribonucleotide excision repair. Further, we review and discuss how ribonucleotide error correction occurs in eukarya, archaea, and bacteria, providing perspectives across a broad range of organisms and ribonucleotide resolution pathways.
Julianna R. Cresti, Lyle A. Simmons· Critical reviews in biochemi...· 0 citations
The “RNA World” hypothesis posits a reasonable scenario for the origin of life. A question central to this scenario is: which RNA species may have emerged as the first ribozyme, thereby initiating the earliest stage of life? To date, experimental and computational studies have focused on ribozymes playing a constructive role in RNA synthesis—such as those facilitating template-directed copying or nucleotide synthesis. However, the answer remains unconvincing, primarily because their functional complexity likely demands lengthy sequences, making de novo emergence improbable. Here, we propose an alternative scenario: a destructive ribozyme, which is potentially quite short, may have emerged first. This ribozyme could cleave other RNA molecules to generate building blocks for its own replication. Notably, the dilemma for a destructive ribozyme is that it may degrade molecules of its own type, thus making it difficult to thrive as an RNA species. Using computational modeling, we demonstrate that an exonuclease ribozyme encoded within a circular RNA genome (thus evading self-attack) can spread within an RNA pool and may subsequently give rise to those constructive ribozymes. Our findings offer a new perspective on the initiation of the RNA world, thereby enriching both theoretical and experimental frameworks for understanding the origin of life.
Abstract Synthetic riboswitches are efficient tools to render gene expression inducible by small molecules. Here, we present a novel riboswitch platform based on RNA triplexes stabilizing non-polyadenylated transcripts while still allowing translation of the respective messenger RNA. By incorporating a tetracycline aptamer, we rendered triplex formation dependent upon the presence of the ligand. The new riboswitch platform was able to induce gene expression up to 25-fold as a standalone switch. Combination of the triplex system with an aptazyme placed into the 5′-UTR achieved a 42-fold induction of gene expression. Further, we were able to exchange the triplex, the aptamer, the maturation platform cleaving off the poly-A tail of the switch platform, and the regulated transgene, demonstrating a high degree of modularity. The new riboswitch platform is designed to function independently of nuclear processes such as transcription and RNA processing, potentially expanding the application of these synthetic riboswitches to strictly cytoplasmic therapeutic RNA vectors.
Pierre Gommeringer, Elisabeth Müller, Sıla Köse et al.· Nucleic Acids Research· 1 citation
circDesign is presented, an algorithm that explicitly incorporates IRES structural deviation into circRNA sequence design while jointly optimizing codon adaptation and thermodynamic stability and establishes IRES structural preservation as a mechanistic design principle for circRNA engineering and position circDesign as a rational framework for therapeutic circRNA development.
Congcong Xu, F. Jiang, Yifan Jiang et al.· Angewandte Chemie· 0 citations
Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.
Jenny J Seo, Chemin Lee, Dongbin Lim et al.· Cell· 0 citations
ADP-ribosylation (ADPr), long recognized as a canonical protein post-translational modification, has recently expanded to include targeting nucleic acids, uncovering a diverse landscape of noncanonical biological functions. Emerging evidence suggests that ADPr at the 5'-phosphate terminus of DNA is implicated in the DNA damage response, yet understanding its precise molecular function has been hampered by the lack of structurally defined chemical probes. Here, we report the stereoselective synthesis of deoxynucleotide-phospho-ADPr (dN-P-ADPr) probes, representing native fragments of terminal DNA-ADPr. Our strategy leverages a mild, stereocontrolled glycosylation to construct the challenging ribosyl-phosphate linkage, followed by P(III)-P(V) coupling to establish the pyrophosphate bridge. This robust toolkit enabled the systematic biochemical profiling of DNA-ADPr hydrolases across diverse kingdoms of life. Remarkably, using these newly developed probes, we uncover hydrolases across the diversity of life capable of reversing ADPr modifications at phosphorylated DNA ends. We further show that these enzymes exhibit an absolute preference for the native-like α-anomer, independent of the identity of the adjacent DNA nucleobase, suggesting that substrate recognition is governed primarily by the ADPr-phosphate linkages rather than the local nucleobase context. Together, these synthetic probes and biochemical insights provide an essential foundation for deciphering the biological landscape of noncanonical ADPr.
Lingxiao Liu, Yang Lu, Zongxing Yu et al.· Journal of the American Chem...· 0 citations
