Cap-related modifications of RNA regulate binding to IFIT proteins.

All cells in our body are equipped with receptors to recognize pathogens and trigger a rapid defense response. As a result, foreign molecules are blocked, and cells are alerted to the danger. Among the many molecules produced in response to viral infection are interferon-induced proteins with tetratricopeptide repeats (IFITs). Their role is to recognize foreign mRNA and eliminate it from the translational pool of transcripts. In the present study, we used biophysical methods to characterize the interactions between the IFIT1 protein and its partners IFIT2 and IFIT3. IFIT1 interacts with IFIT3 with nanomolar binding affinity, which did not change significantly in the presence of the preformed IFIT2/3 complex. The interactions between IFIT2 and IFIT3 and IFIT1 and IFIT2 were one order of magnitude weaker. We also present kinetic data of the interactions between the IFIT protein complex and short RNA bearing various modifications at the 5' end. We show kinetic parameters for interaction between the IFIT complex and RNA with m6Am modification. The results show that the cap-adjacent m6Am modification is a stronger signature than cap1 alone. It blocks the formation of a complex between IFIT proteins and m7Gpppm6Am-RNA much more effectively than other cap modifications. In contrast, m6A in the 5'UTR is not recognized by IFIT proteins and does not contribute to translation repression by IFIT proteins. The data obtained are important for understanding the regulation of expression of genetic information. They indicate that 2'-O and m6Am modifications modulate the availability of mRNA molecules for proteins of innate immune response.

N2 modified dinucleotide cap analogs as a potent tool for mRNA engineering.

mRNA-based vaccines are relatively new technologies that have been in the field of interest of research centers and pharmaceutical companies in recent years. Such therapeutics are an attractive alternative for DNA-based vaccines since they provide material that can be used with no risk of genomic integration. Additionally, mRNA can be quite easily engineered to introduce modifications for different applications or to modulate its properties, for example, to increase translational efficiency or stability, which is not available for DNA vectors. Here, we describe the use of N2 modified dinucleotide cap analogs as components of mRNA transcripts. The compounds obtained showed very promising biological properties while incorporated into mRNA. The presented N2-guanine modifications within the cap structure ensure proper attachment of the dinucleotide to the transcripts in the IVT reaction, guarantees their incorporation only in the correct orientation, and enables highly efficient translation of mRNA both in the in vitro translation system and in human HEK293 cells.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

miR-483-5p orchestrates the initiation of protein synthesis by facilitating the decrease in phosphorylated Ser209eIF4E and 4E-BP1 levels.

Eukaryotic initiation factor 4E (eIF4E) is a pivotal protein involved in the regulatory mechanism for global protein synthesis in both physiological and pathological conditions. MicroRNAs (miRNAs) play a significant role in regulating gene expression by targeting mRNA. However, the ability of miRNAs to regulate eIF4E and its phosphorylation remains relatively unknown. In this study, we predicted and experimentally verified targets for miR-483-5p, including eukaryotic translation initiation factor eIF4E and its binding proteins, 4E-BPs, that regulate protein synthesis. Using the Web of Science database, we identified 28 experimentally verified miR-483-5p targets, and by the TargetScan database, we found 1818 predicted mRNA targets, including EIF4E, EIF4EBP1, and EIF4EBP2. We verified that miR-483-5p significantly reduced ERK1 and MKNK1 mRNA levels in HEK293 cells. Furthermore, we discovered that miR-483-5p suppressed EIF4EBP1 and EIF4EBP2, but not EIF4E. Finally, we found that miR-483-5p reduced the level of phosphorylated eIF4E (pSer209eIF4E) but not total eIF4E. In conclusion, our study suggests that miR-483-5p's multi-targeting effect on the ERK1/ MKNK1 axis modulates the phosphorylation state of eIF4E. Unlike siRNA, miRNA can have multiple targets in the pathway, and thereby exploring the role of miR-483-5p in various cancer models may uncover therapeutic options.

Mesenchymal stem cell engineering by ARCA analog-capped mRNA.

We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. ß-S-ARCA D1 and ß-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that ß-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based in vitro transfection of HPLC-purified and ARCA- or ß-S-ARCA D1-capped mRNA is optimal for MSC engineering.

The potential of N2-modified cap analogues for precise genetic manipulation through mRNA engineering.

The technology of mRNA-based drugs is currently being intensively developed and implemented. Medical products of this type are already being used as viral vaccines and could potentially find application in a wide range of diseases. The tremendous interest in mRNA is due to the relatively easy production process, which can be quickly adapted to meet societal needs. The properties of this molecule depend on the structure of its individual components, such as the structure of the cap at the 5' end. Modifications of the cap significantly affect the translational potential and lifespan of the whole mRNA. In the current work, we present the synthesis of derivatives of cap analogues modified at the N2 position of 7-methylguanosine. In addition to the substituent at the N2 position, the derivatives had either an extended triphosphate chain, a thiophosphate modification, an added cap1-modified nucleotide or an extended linker between the substituent and 7-methylguanosine. The compounds were tested for use as translation inhibitors and as components for mRNA preparation and appeared of interest for both applications.

RNA sensor response in HeLa cells for transfected mRNAs prepared in vitro by SP6 and HiT7 RNA polymerases: A comparative study.

In vitro transcribed (IVT) synthetic mRNAs are in high demand due to their attractive bench to clinic translational processes. Mainly, the procedure to make IVT mRNA using bacteriophage RNA polymerases (RNAP) is relatively uncomplicated and scalable to produce large quantities in a short time period. However, IVT mRNA preparations are accompanied by contaminants such as double-stranded RNA (dsRNA) as by-products that elicit undesired cellular immune responses upon transfections. Therefore, removing dsRNA contaminants is critical in IVT mRNA preparations for therapeutic applications. One such method to minimize dsRNA contaminants is to use genetically modified thermostable bacteriophage polymerase, HiT7 RNAP that performs IVT reaction at a higher temperature than typically used. However, the cellular RNA sensor response for IVT mRNA preparations by HiT7 RNAP is not characterized. Here, we compared the cellular RNA sensor response for mRNAs prepared by HiT7 RNAP (at 50°C) and SP6 RNAP (at 37°C) in HeLa cells. We show that IVT mRNA preparations by HiT7 RNAP reduced the dsRNA levels and dsRNA specific RNA sensor response (retinoic acid-inducible gene I, RIG-I and melanoma differentiation-associated 5, MDA5) compared to the IVT mRNA preparations by SP6 RNAP. Similarly, the incorporation of pseudouridine nucleotides instead of uridine nucleotides reduced dsRNA sensor response and increased the mRNA translation. Overall, the least dsRNA mediated RNA sensor response is observed when mRNA is synthesized by HiT7 RNAP and incorporated with pseudouridine nucleotides.

Studies on the anti-hepatitis C virus activity of newly synthesized tropolone derivatives: identification of NS3 helicase inhibitors that specifically inhibit subgenomic HCV replication.

We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC50=3.4 microM), 3,5,7-tri[(4'-methylpiperazin-1'-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9 microM (EC50) and exhibited the lowest cytotoxicity (CC50)>1 mM resulting in a selectivity index (SI=CC50/EC50)>21. The most efficient replication inhibitor, 3,5,7-tri[(4'-methylpiperidin-1'-yl)methyl]tropolone (6), inhibited RNA replication with an EC50 of 32.0 microM and a SI value of 17.4, whereas 3,5,7-tri[(3'-methylpiperidin-1'-yl)methyl]tropolone (7) exhibited a slightly lower activity with an EC50 of 35.6 microM and a SI of 9.8.

Circular dichroism analysis for multidomain proteins: studies of the irreversible unfolding of Hepatitis C virus helicase.

The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) is a bifunctional enzyme with RNA-dependent NTPase/RNA helicase and serine protease activities, and thus represents a promising target for anti-HCV therapy. These functions are performed by two distinct moieties; the N-terminal protease domain and the C-terminal helicase domain that further folds into three structural subdomains. To obtain lower molecular mass proteins suitable for nuclear magnetic resonance studies of helicase-inhibitor complexes, helicase domains 1, 2, and 1+2 devoid of a hydrophobic beta-loop were overexpressed and purified. Circular dichroism studies were carried out to confirm the secondary structure content and to determine thermodynamic parameters describing the stability of the proteins. Both thermal and GuHCl-induced unfolding experiments confirmed the multidomain organization of the helicase. The unfolding transition observed for domain 1+2 was in agreement with the model of two well-resolved successive steps corresponding to the independent unfolding of domains 1 and 2, respectively. In the case of the full-length helicase, the presence of domain 3 remarkably changed the transition profile, leading to fast and irreversible transformation of partially unfolded protein.

New acridone-4-carboxylic acid derivatives as potential inhibitors of hepatitis C virus infection.

A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase activity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C virus (HCV). The compounds were also tested as putative transcription inhibitors of in vitro transcription based on the DNA-dependent T7 RNA polymerase. Most of the acridone derivatives tested were transcription inhibitors; however, only four of them inhibited the NS3 helicase at low concentrations (IC(50) from 3 microM to 20 microM) and were therefore selected for further studies on the mechanism of inhibition. The acridone derivatives probably act via intercalation into double-stranded nucleic acids but they may also interact directly with viral enzymes. Selected carboxamides were tested in the subgenomic HCV replicon system. Two of the compounds: N-(pyridin-4-yl)-amide and N-(pyridin-2-yl)-amide of acridone-4-carboxylic acid are efficient RNA replication inhibitors with selectivity indexes of 19.4 and 40.5, respectively, proving that the acridone derivatives may be regarded as potential antiviral agents.