A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

The artificial control of DNA structure and function is an attractive field in chemical and synthetic biology, [1] and light is a powerful and convenient trigger: it is non-invasive, provides high spatio-temporal resolution, and offers the option of orthogonality. DNA is reactive to light: the UVlight-induced cyclodimerization of pyrimidine nucleosides is an important type of DNA damage and has been studied intensively. [2] Interestingly, this chemical property has never been exploited for the construction of DNA-based reversible photoswitches, and all published work in this field is based on the covalent functionalization of DNA with small autonomous photoactive molecules. [3] Different classes of such photoactive molecules have been studied for DNA photoregulation; [4] azobenzenes have been used most frequently and were, for example, employed for modulating oligonucleotide duplex and triplex formation and DNA transcription. [5] Other substance classes studied in this context include arylvinyl derivatives, [6] spiropyranes, [3a] and recently also diarylethenes. [3a, 7] While a certain influence of photoisomerization on the properties of the nucleic acid was always observed, these approaches share one common limitation: because an autonomous photoswitch was attached to the DNA, either as an appendage or substituting for nucleosides, the rearrangement of chemical bonds upon encountering a photon (i.e., the photochemical reaction) was strictly confined to this non-nucleosidic moiety.Recently, our lab reported a new type of diarylethene photoswitches in which one of the two aryl moieties was replaced by a nucleoside, namely 7-deaza-8-methyldeoxyadenosine (Scheme 1). [8] These photoswitches were synthesized in a convergent multi-step approach in which a substi-tuted cyclopentenyl boronic ester was reacted with protected 7-iodo-8-methyl-7-deazadeoxyadenosine by Suzuki crosscoupling, followed by deprotection. Upon irradiation with light, these compounds were found to undergo a highly efficient, reversible, electrocyclic rearrangement and the switching wavelength could be tuned by the chemical nature of substituents. Switching was found to be near-quantitative in aprotic solvents, and the compounds retained the key properties of nucleotides, such as their capability to form Watson-Crick base-pairs. Unfortunately, the photoisomerization was found to proceed with low efficiency in aqueous solvents, and the demanding synthesis involved limited the application of these photoswitches to oligonucleotides.To develop straight-forward access to truly photoswitchable DNA, we reconsidered our design approach; in contrast to 7-iodo-8-methyl-7-deazadeoxyadenosine, the 5-iodo-substituted pyrimidine nucleosides 5I-dU and 5I-dC represent oligonucleotide modifications readily available from commercial suppliers, and offer the desired reactivity for different cross-coupling reactions. [9] This could allow for the postsynthetic conversion of an iodo-modified oligonucleotide into a photoswitch, leading to the target compounds shown i...

It has been more than 50 years since the discovery of dinucleoside polyphosphates (Np n Ns) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated Np n Ns serve as RNA caps in Escherichia coli. Np n Ns are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5′-pyrophosphohydrolase (RppH) and bis(5′-nucleosyl)-tetraphosphatase (ApaH) are able to remove the Np n N-caps from RNA. ApaH is able to cleave all Np n N-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as Np n Ns are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.

Single‐step aqueous cross‐coupling reactions of nucleobase‐halogenated 2′‐deoxynucleosides (8‐bromo‐2′‐deoxyadenosine, 7‐iodo‐7‐deaza‐2′‐deoxyadenosine, or 5‐iodo‐2′‐deoxy‐uridine) or their 5′‐triphosphates with 4‐boronophenylalanine or 4‐ethynylphenylalanine have been developed and used for efficient synthesis of modified 2′‐deoxynucleoside triphosphates (dNTPs) bearing amino acid groups. These dNTPs were then tested as substrates for DNA polymerases for construction of functionalized DNA through primer extension and PCR. While 8‐substituted adenosine triphosphates were poor substrates for DNA polymerases, the corresponding 7‐substituted 7‐deazaadenine and 5‐substituted uracil nucleotides were efficiently incorporated in place of dATP or dTTP, respectively, by Pwo (Pyrococcus woesei) DNA polymerase. Nucleotides bearing the amino acid connected through the less bulky acetylene linker were incorporated more efficiently than those directly linked through a more bulky phenylene group. In addition, combinations of modified dATPs and dTTPs were incorporated by Pwo polymerase. Novel functionalized DNA duplexes bearing amino acid moieties were prepared by this two‐step approach. PCR can be used for amplification of duplexes bearing large number of modifications, while primer extension is suitable for introduction of just one or several modifications in a single DNA strand.

Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in ∼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.

Primer extension is used to incorporate labeled nucleoside triphosphates into oligonucleotides (ONs). Aminophenyl and nitrophenyl modifications serve as electrochemical labels that are detectable by either oxidation or reduction, respectively (see representation of the ON double strand and redox curves). The redox potentials of the labels differ depending on the nucleobase and respond to incorporation into ONs.

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.

Durch Primerverlängerung wurden markierte Nucleosidtriphosphate in Oligonucleotide (ONs) eingebaut. Aminophenyl‐ und Nitrophenylmodifikationen dienen als elektrochemische Markierungen, die oxidiert bzw. reduziert werden können (siehe Darstellung eines ON‐Doppelstrangs und Redoxkurven). Die Redoxpotentiale der Markierungen unterscheiden sich je nach Nucleobase und ändern sich beim Einbau in ONs.