The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of phosphodiester backbone RNA has been incompletely understood. There is experimental evidence for an important role adenine (A38) and a guanine (G8), it proposed that these act in general acid–base catalysis. In this work we show large reduction rate on substitution A38 purine (A38P) can be reversed replacement 5′-oxygen atom at scissile phosphate sulfur (5′-PS), much better leaving group. This...

We describe the multifactorial origins of catalysis by twister ribozyme. provide evidence that adenine immediately 3′ to scissile phosphate (A1) acts as a general acid. Substitution ring nitrogen atoms indicates very unusually N3 A1 is proton donor oxyanion leaving group. accommodated in specific binding pocket raises its pKa toward neutrality, juxtaposes with O5′ be protonated, and helps create in-line trajectory required for nucleophilic attack. performs acid while G33 base. A 100-fold...

The preparation of highly modified mRNAs and visualization their cellular distribution are challenging. We report in-cell application in vitro transcribed mRNA containing natural base modifications site-specifically introduced artificial nucleotides. Click chemistry on allows cells with excellent signal intensities. While non-specific introduction reporter groups often leads to loss functionality, we combined the benefits from site-specificity 3'-UTR incorporated unnatural nucleotides...

An unnatural nucleotide capable of Diels–Alder cycloaddition is site-specifically introduced into RNA <italic>via</italic> T7 <italic>in vitro</italic> transcription.

Inverse electron demand Diels-Alder cycloadditions are extremely useful tools for orthogonal labeling of biomolecules such as proteins or small molecules in a cellular context. In-cell dienophile-modified RNA oligonucleotides using cycloaddition reactions has not been demonstrated before. In this study we report site-specific modified with norbornene derivatives at predefined sequence position within an vitro and mammalian cells various tetrazine-fluorophore conjugates. The approach could...

Site-specific RNA modification with methyl cyclopropene moieties is performed by T7 in vitro transcription. An existing unnatural base functionalized a moiety and used transcription reactions to produce site-specifically cyclopropene-modified molecules. The posttranscriptional inverse electron demand Diels-Alder cycloaddition reaction selected tetrazine-fluorophore conjugate demonstrated.

Protoporphyrin (PP) and biliverdin (BV) give rise to the enormous diversity in avian egg coloration. Egg color serves several ecological purposes, including post-mating signaling camouflage. camouflage represents a major character of open-nesting birds which accomplish protection their unhatched offspring against visually oriented predators by cryptic Cryptic coloration evolved match predominant shades found nesting environment. Such selection pressure for evolution colored or eggs should be...

We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3

Nucleic acid sensing is crucial for advancing diagnostics, therapeutic monitoring and molecu-lar biology research, by enabling the precise identification of DNA RNA interactions. Here, we present an innovative platform based on DNA-functionalized whispering gallery mode (WGM) microlasers. By correlating spectral shifts in laser emission to changes refractive index, demonstrate real-time detection hybridization structural changes. The addition gold nanoparticles strands significantly enhances...

Nucleic acid sensing is crucial for advancing diagnostics, therapeutic monitoring, and molecular biology research by enabling the precise identification of DNA RNA interactions. Here, we present an innovative platform based on DNA-functionalized whispering gallery mode (WGM) microlasers. By correlating spectral shifts in laser emission to changes refractive index, demonstrate real-time detection hybridization structural changes. The addition gold nanoparticles strands significantly enhances...

Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA RNA synthesis. These are consequently strong replication blocks. In addition adducts give rise to point frameshift mutations. The translesion synthesis (TLS) polymerase η is able bypass slowly adduct such as the widely studied 2-aminofluorene-dG its acetylated analogue mainly an error-free manner. Replicative polymerases contrast fully blocked by lesion. Here, we show that TLS efficiency of...

Flipped out: The cisplatin–(1,3-GTG) adduct, a strongly cytotoxic DNA lesion generated by the action of cisplatin therapeutics, is partially bypassed Y-family polymerase η. Crystal structure data on this DNA–protein complex explain partial-bypass process. central dT unit turned out and blocks movement enzyme along duplex. Detailed facts importance to specialist readers are published as "Supporting Information". Such documents peer-reviewed, but not copy-edited or typeset. They made available...

Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules influenced the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural possible, possibly within cells. Site-directed spin-labeling of nitroxides in combination with, for example, pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy has been shown provide such information. However, in-cell measurements, usually used...

Xeno nucleic acids (XNAs) constitute a class of synthetic acid analogues characterized by distinct, non-natural modifications within the tripartite structure polymers. While most described XNAs contain modification in only one structural element scaffold, this work explores XNA chemical space to create more divergent variants with multiple parts nucleosidic scaffold. Combining enhanced nuclease resistance α-l-threofuranosyl (TNA) and almost natural-like replication efficiency fidelity...

Spin labeling of<italic>in vitro</italic>transcribed RNA by iEDDA click chemistry is demonstrated. This allows the determination of distance distributions between two nitroxide spin labels PELDOR in a self-complementary duplex.

Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those expand chemical scope DNA RNA, thus, could enable evolution novel aptamers or ribozymes by vitro selection (systematic ligands exponential enrichment, SELEX). However, application such reverse transcription (rtc), which is a key step for RNA-based SELEX, has not been reported so far. implication Romesberg's...

Frameshift mutations: Bulky adduct DNA lesions, such as the C8-acetylaminofluorene dG lesion (AAF-dG), induce frameshift mutations if they are placed in special gene sequences. With help of synthetic AAF-dG lesions inserted into different repetitive frameshift-prone sequences, molecular basis −I, −II, and −III frameshifting events was determined.