RNA-based therapeutics have emerged as a powerful class of drugs, highlighted by the rapid development and success of COVID-19 vaccines. Therapeutic RNA synthesis relies on in vitro transcription (IVT), most commonly using bacteriophage RNA polymerases (RNAP) such as T7 RNAP and SP6 RNAP. However, efficient incorporation of modified nucleotides and the reduction of double-stranded RNA (dsRNA) by-products to improve pharmacokinetic properties and reduce immunostimulatory effects remain major challenges. While T7 RNAP has been extensively engineered to expand substrate tolerance and reduce dsRNA formation, engineering efforts for SP6 RNAP remain comparatively limited, and a direct functional comparison between the two enzymes is lacking. Here, we systematically compared wild-type (wt) T7 and SP6 RNAP with respect to nucleotide analogue incorporation and dsRNA formation. We evaluated engineered variants of both polymerases for their ability to incorporate the modified nucleotides 2'‑F‑UTP and 2'‑O‑methyl‑UTP during IVT. In general, T7 RNAP displayed higher yields than SP6 RNAP for long RNA transcripts and a T7 RNAP variant demonstrated the highest acceptance of 2'‑F‑UTP. However, when using 2'‑OMe‑UTP in IVT, only a SP6 RNAP enabled synthesis of a ~ 760 nt long transcript. Moreover, the assessment of dsRNA formation with both wild-type polymerases revealed that SP6 RNAP produced substantially less dsRNA than T7 RNAP during IVT. Together, these results highlight distinct and complementary strengths of T7 and SP6 RNAP. While the SP6 RNAP FA variant showed particular promise for the synthesis of mRNA containing bulky nucleotide modifications, the reduced dsRNA formation observed for SP6 RNAP wt suggests an additional advantage for improving RNA quality.

Lysine acetylation plays a prominent regulatory role in eukaryotic cells. Yet, determining the functional consequences of acetylation for a given protein represents a considerable challenge. For instance, lysine residues are subject to various posttranslational modifications, rendering interpretation of mutational studies difficult. The genetic code expansion technology enables site-specific incorporation of acetyllysine (AcK) into proteins, but the applicability of AcK is limited, as within cells, the acetyl group is removed by deacetylases. Here, we show that site-specific incorporation of the non-hydrolyzable AcK analog ketolysine (KeK) into ubiquitin closely resembles the structural and functional effects of AcK incorporation. Furthermore, AcK and KeK can be efficiently incorporated into the tumor suppressor p53 in cells. However, whereas AcK becomes deacetylated, KeK remains stable. Accordingly, incorporation of KeK, but not AcK, affects p53-mediated transcription. Thus, we propose that KeK is a well-suited AcK surrogate for studying acetylation of a given protein in cells.

1,5-Anhydrohexitol nucleic acid (HNA) is a promising xeno nucleic acid (XNA) for applications such as aptamers and catalysts, due to its favourable physico-chemical properties. Realizing this potential requires efficient and high-fidelity polymerases capable of processing HNA. A key component are HNA reverse transcriptases that convert HNA into DNA, an essential step in standard SELEX workflows. Although HNA reverse transcriptases have been generated by directed evolution, structural insight is essential to guide further enzyme engineering. Here, we report the 2.8 Å crystal structure of the engineered HNA reverse transcriptase KOD-H4, derived from the B-family DNA polymerase of Thermococcus kodakarensis, captured in a closed ternary complex with dATP, a 3'-terminated primer and a mixed HNA/DNA template. Compared to a previously reported open ternary KOD-H4 structure, the presented structure adopts a more closed conformation with increased finger and thumb domain closure and formation of a canonical Watson-Crick-Franklin base pair at the insertion site. Direct downstream nucleotides show more distorted base pairing and one HNA residue transits from the unusual 1C4 conformation it adopted in the open complex to the 4C1 hexitol sugar conformation. These findings demonstrate that KOD-H4 can form a closed, pre-catalytic complex resembling that of the wildtype enzyme with natural substrates, and reveal state-dependent conformational flexibility of HNA. Such flexibility should be considered in the design and optimization of enzymes that process HNA.

Human RNA ligase 1 (Rlig1) is a recently identified human 5 '-3 ' RNA ligase required for maintaining 28S ribosomal RNA integrity and promoting cell survival under oxidative stress. Although its enzymatic activity suggests a role in RNA processing and repair, the broader molecular context of Rlig1 remains poorly defined. Here, we identified potential Rlig1-associated proteins by affinity enrichment-mass spectrometry. Subsequent analysis revealed proteins involved in RNA surveillance and processing, including RNA-binding and end-processing enzymes, and indicated strong enrichment of ribosomal proteins. We showed that Rlig1 interacts with 80S ribosomes in vitro. Consistent with this observation, polysome profiling revealed recruitment of Rlig1 to ribosomal fractions under oxidative stress. Functionally, Rlig1-knockout (KO) HEK293 cells exhibited accelerated polysome loss and significantly reduced global protein synthesis compared to wild-type (WT) HEK293 cells during oxidative stress. In addition, we showed that stress-induced RNA fragments containing a 5 '-PO4 end accumulated in Rlig1-KO cells. Among these, transfer RNA halves were prominently enriched. Together, our study links Rlig1 to ribosomal complexes and suggests that Rlig1 contributes to preserving RNA integrity and supporting translational capacity during oxidative stress.

Bacteriophage T7 RNA polymerase (T7 RNAP) is a key enzyme for in vitro transcription (IVT) and plays a central role in the production of synthetic mRNA for research and therapeutic applications. However, IVT frequently generates double-stranded RNA (dsRNA) as an undesired by-product, which can trigger innate immune responses and compromise mRNA quality. Increasing reaction temperatures reduces dsRNA formation, however, the wild-type T7 RNA polymerase exhibits limited stability under such conditions. Therefore, polymerase variants with enhanced thermotolerance enable more robust transcription at elevated temperatures while minimizing dsRNA generation. To address this limitation, we aimed at obtaining T7 RNA polymerase variants with increased thermotolerance using the Protein Repair One Stop Shop (PROSS) web server. Four crystal structures of T7 RNA polymerase, comprising a promoter complex, an initiation complex, and two elongation complexes were used as input for independent PROSS runs. Mutations shared across all four designs for each PROSS index were then combined to generate multi-structure PROSS Combined Designs (PCDs). In the subset evaluated experimentally, PCD9 retained full-length transcription activity at temperatures up to 48 °C, whereas wild-type T7 RNA polymerase showed strong loss of activity under the same buffer conditions. At 48 °C, PCD9 supported the synthesis of kilobase-length scale transcripts and produced no detectable dsRNA signal in a dot blot assay. In contrast, the wild-type enzyme generated strong dsRNA signals at 37 °C and failed to produce detectable RNA at 48 °C. Additional PROSS variants derived exclusively from the elongation complex structure were inactive at both 37 °C and 48 °C. Together, these results show that multi-structure PROSS design can yield a thermotolerant T7 RNA polymerase with improved performance at elevated temperature and reduced dsRNA byproduct formation. The findings also suggest that restricting stability design to a single structural state may not fully capture the requirements of a highly dynamic enzyme.

The ubiquitin (Ub) ligase E6AP, encoded by the UBE3A gene, has been causally associated with human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease-relevant substrates of E6AP is still limited, presumably because at least some of these interactions are rather transient, a phenomenon observed for many enzyme-substrate interactions. Here, we introduce a novel approach to trap such potential transient interactions by combining a stable E6AP-Ub conjugate mimicking the active state of this enzyme with photo-crosslinking (PCL) followed by affinity enrichment coupled to mass spectrometry (AE-MS). To enable PCL, we equipped Ub with diazirine moieties at distinct positions. We validated our PCL assisted AE-MS approach by identification of known (e. g. PSMD4, UCHL5) and potential new (e. g. MSH2) substrates of E6AP. Our findings suggest that PCL assisted AE-MS is indeed suited to identify substrates of E6AP, thereby providing insights into E6AP-associated pathologies, and, potentially, of other enzymes of the Ub-conjugating system.

Xeno nucleic acids (XNAs) are unnatural analogues of the natural nucleic acids in which the canonical ribose or deoxyribose rings are replaced with alternative sugars, congener structures or even open-ring configurations. The expanding repertoire of XNAs holds significant promise for diverse applications in molecular biology as well as diagnostics and therapeutics. Key advantages of XNAs over natural nucleic acids include their enhanced biostability, superior target affinity and (in some cases) catalytic activity. Natural systems generally lack the mechanisms to transcribe, reverse transcribe or replicate XNAs. This limitation has been overcome through the directed evolution of nucleic acid-modifying enzymes, especially polymerases (pols) and reverse transcriptases (RTs). Despite these advances, the mechanisms by which synthetic RT enzymes read these artificial genetic polymers remain largely unexplored, primarily due to a scarcity of structural information. This study unveils first structural insights into an evolved thermostable DNA pol interacting with the XNA 1,5-anhydrohexitol nucleic acid (HNA), revealing unprecedented HNA nucleotide conformations within a ternary complex with the enzyme. These findings not only deepen our understanding of HNA to DNA reverse transcription but also set the stage for future advancements of this and similar enzymes through deliberate design.

PARP7, a mono-ADP-ribosyl (MAR) transferase, is a key suppressor of the type I interferon (IFN-I) IFNβ in various tumor cells and a validated drug target. This negative regulation is reversed by small-molecule inhibitors of PARP7 catalytic activity, resulting in increased IFN-β expression. Yet, the mechanism of action of PARP7 inhibitors remains unclear because the relevant substrates of PARP7-mediated MARylation are unknown. Using an optimized analog- sensitive chemical genetic (ASCG) approach, we identified the co-activators, p300 and CBP, as nuclear PARP7 substrates. We identified an α-helical domain in PARP7 essential for p300/CBP interaction, MARylation, and proteasome degradation. Disrupting PARP7-p300/CBP interaction prevents PARP7's suppression of IFNβ in colorectal cancer cells. p300/CBP reciprocally regulate PARP7's activity and nuclear localization. Intriguingly, treatment with PARP7 inhibitors increased IFNβ expression more than PARP7 knockout in a p300/CBP-dependent manner. Our findings suggest that in some contexts, IFNβ induction by PARP7 inhibitors occurs via two mechanisms: inhibiting MARylation of p300/CBP (loss-of-function) and stabilizing the PARP7- p300/CBP complex (gain-of-function). Teaser:Chemical genetics discovery of p300 and CBP as substrates of PARP7 that are essential for PARP7-mediated regulation of IFNβ via a dual mechanism.

We report on the development of inhibitors of human RNA ligase 1 (Rlig1).

RNA ligases are essential for the repair, splicing, and editing of RNA across various biological systems. Recently, a new enzyme that catalyses 5’-3’ RNA ligation – RNA ligase 1 (Rlig1) – was identified in vitro . However, the in vivo biological functions of Rlig1 have remained elusive. Here, we reveal the role of Rlig1 during vertebrate development using embryonic and larval zebrafish as a model system. We found that rlig1 mRNA is maternally deposited and present ubiquitously during early embryogenesis, whereas at larval stages it localises to the brain and eyes. Interestingly, CRISPR/Cas9-generated rlig1 knockout zebrafish exhibited no overt morphological abnormalities, but showed reduced behavioural responsiveness to visual stimuli along with massively perturbed transcriptomes and widespread dysregulation of core metabolic and translational pathways. Brain-wide calcium imaging in rlig1 knockout larvae revealed decreased neuronal activity in key regions for visual processing, consistent with the observed behavioural defects. Together, our findings identify a role for Rlig1 in maintaining the integrity and function of the nervous system and uncover a new link between neuronal RNA processing, development, and sensory-motor computation.

Dinucleoside polyphosphates (NpnNs) are known as alarmones but their functions in cellular metabolism remain largely unexplored till to date. Here, we report new data concerning their cellular quantification using mass spectrometry-based methods. Key for this approach were 13C-isotope-labeled internal standards of eight different compounds (13C-ApnN, n = 3,4; N = Adenosine, Cytidine, Guanosine, Uridine) that were chemically synthesized from 13C5-adenosine. For this, a novel synthesis strategy was developed. These compounds were used to account for losses during the extraction for the determination of intracellular Ap3/4N-levels. Cell samples from two human cell lines, HEK293T and H1299, were measured using a triple quad mass spectrometer (TQ-MS). Additionally, menadione was added to the cell dishes to generate oxidative stress. We were able to reproduce previous findings that all Ap3/4N levels increase in stressed cells. In addition, we showed that cells lacking the Np3N hydrolase Fhit (fragile histidine triad) (H1299, FHIT-negative) exhibit hundred-fold increased levels of Ap3Ns but also ten-fold increased levels of Ap4Ns. This finding contradicts previous data, where no impact of the expression of Fhit on Ap4N-levels was detected. For FHIT-negative cells, no significant increase in Ap3/4N levels was observed when oxidative stress was applied, suggesting that a change in hydrolase activity could be the primary stress response rather than increased biosynthesis.

Reverse transcription polymerase chain reaction (RT-PCR) has evolved as a widely used approach in biotechnology and molecular diagnostics. It represents a powerful tool for amplifying and analysing RNA molecules and has therefore found widespread applications in profiling gene expression, viral detection and the diagnosis of various diseases. Wellestablished methodologies use viral reverse transcriptases (RTs) to transcribe RNA to cDNA and thermostable DNA polymerases (DNA pols) to amplify the resulting target sequence by PCR. This study reports on the development of novel Thermus aquaticus DNA polymerase I (Taq pol) variants that each are able to catalyse both steps simultaneously in a single tube without the need of viral RTs. In combination with their excellent thermostability (up to 95 °C), the novel Taq pol variants are suitable for employment in dye- or probe-based RNA detection methods. Moreover, the herein reported Taq pol variants are capable of performing multiplex detection of various RNA targets in a single tube with a single enzyme. Thus, discovery marks a significant advancement of current RT-PCR approaches and contributes simplifying and reducing costs in molecular diagnostics.

ADP-ribosylation is a post-translational modification catalyzed by the enzyme family of polyadenosine diphosphate (ADP)-ribose) polymerases (PARPs). This enzymatic process involves the transfer of single or multiple ADP-ribose molecules onto proteins, utilizing nicotinamide adenine dinucleotide (NAD+ ) as a substrate. It, thus, plays a pivotal role in regulating various biological processes. Unveiling PARP-selective protein targets is crucial for a better understanding of their biological functions. Nonetheless, this task proves challenging due to overlapping targets shared among PARP family members. Therefore, we applied the "bump-and-hole" strategy to modify the nicotinamide binding site of PARP1 by introducing a hydrophobic pocket ("hole"). This PARP1-mutant binds an orthogonal NAD+ (Et-DTB-NAD+ ) containing an ethyl group ("bump") at the nicotinamide moiety. Furthermore, we added a desthiobiotin (DTB) tag directly to the adenosine moiety, enabling affinity enrichment of ADP-ribosylated proteins. Employing this approach, we successfully identified protein targets modified by PARP1 in cell lysate. This strategy expands the arsenal of chemically modified NAD+ analogs available for studying ADP-ribosylation, providing a powerful tool to study these critical post-translational modifications.

Post-translational modifications (PTMs) of histones have fundamental effects on chromatin structure and function. While the impact of PTMs on the function of core histones are increasingly well understood, this is much less the case for modifications of linker histone H1, which is at least in part due to a lack of proper tools. In this work, we establish the assembly of intact chromatosomes containing site-specifically ubiquitylated and acetylated linker histone H1.2 variants obtained by a combination of chemical biology approaches. We then use these complexes in a tailored affinity enrichment mass spectrometry workflow to identify and comprehensively characterize chromatosome-specific cellular interactomes and the impact of site-specific linker histone modifications on a proteome-wide scale. We validate and benchmark our approach by western-blotting and by confirming the involvement of chromatin-bound H1.2 in the recruitment of proteins involved in DNA double-strand break repair using an in vitro ligation assay. We relate our data to previous work and in particular compare it to data on modification-specific interaction partners of free H1. Taken together, our data supports the role of chromatin-bound H1 as a regulatory protein with distinct functions beyond DNA compaction and constitutes an important resource for future investigations of histone epigenetic modifications.

Posttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene-encoded variations. One intriguing PTM is ADP-ribosylation, which utilizes nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell-permeable NAD+ analogs and demonstrate their utility for investigating cellular ADP-ribosylation. Using a desthiobiotin-labelled analog for affinity enrichment of proteins that are ADP-ribosylated in living cells under oxidative stress, we identified protein targets associated with host-virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP-ribosylome studies.

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2'-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes.

AbstractEine Dysregulation der DNA‐Methylierung wird mit der Entstehung humaner Krankheiten, insbesondere Krebs, in Verbindung gebracht. Die Untersuchung abnormaler Methylierungsmuster ist daher vielversprechend für die klinische Diagnostik. DNA‐Polymerasen unterscheiden jedoch nicht effektiv zwischen der Umsetzung von 5‐Methylcytosin (5 mC) und unmethyliertem Cytosin, wodurch die Methylierungsinformation während der Amplifikation oder Sequenzierung verloren geht. Aktuelle Nachweismethoden erfordern daher mehrstufige DNA‐Konversionsbehandlungen oder eine sorgfältige Analyse der Sequenzierungsdaten, um einzelne 5 mC‐Basen zu entschlüsseln. Um diese Herausforderung zu überwinden, schlagen wir einen neuartigen DNA‐Polymerase‐vermittelten Ansatz zur Detektion von 5 mC vor. Hier beschreiben wir die Entwicklung einer thermostabilen DNA‐Polymerase‐Variante aus Thermus aquaticus, die eine veränderte Genauigkeit für 5 mC aufweist. Durch einen Screening‐Ansatz konnten wir eine DNA‐Polymerase identifizieren, die während der DNA‐Synthese einen erhöhten Fehleinbau gegenüber 5 mC zeigt. Diese DNA‐Polymerase erzeugt Mutationssignaturen an methylierten CpG‐Stellen, die einen direkten Nachweis von 5 mC durch das Auslesen einer erhöhten Fehlerrate nach der Sequenzierung ohne vorherige Behandlung der Proben‐DNA erlaubt.

Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5-methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi-step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase-mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening-based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.

Rlig1 is the first RNA ligase identified in humans utilising a classical 5 '-3 ' ligation mechanism. It is a conserved enzyme in all vertebrates and is mutated in various cancers. During our initial research on Rlig1, we observed that Rlig1-knockout (KO) HEK293 cells are more sensitive to the stress induced by menadione than their WT counterpart, representing a type of chemical synthetic lethality. To gain further insight into the biological pathways in which Rlig1 may be involved, we aimed at identifying new synthetically lethal small molecules. To this end, we conducted a high-throughput screening with a compound library comprising over 13 000 bioactive small molecules. This approach led to the identification of compounds that exhibited synthetic lethality in combination with Rlig1-KO. In addition to the aforementioned novel compounds that diverge structurally from menadione, we also tested multiple small molecules containing a naphthoquinone scaffold. Six unique compounds were found to be synthetically lethal in combination with Rlig1-KO in HEK293 cells. In addition, several naphthoquinones, which are similar to menadione, were evaluated in this context.