Ribose Research Articles

Facile Synthesis of Valsartan and Ribose Prodrug and Studying its Influence on the Pancreatic Lipase

Valsartan is a widely used drug that belongs to the type of angiotensin receptor blockers generally specified to control high blood pressure (hypertension) and heart failure. In the present work, a new ester prodrug utilizes ribose sugar as a carrier for the valsartan through an esterification reaction. The synthesized compound was characterized by the FT-IR and 1H NMR spectroscopic. The investigation furthermore involved measuring the activity of the lipase enzyme in the blood serum of individuals with high blood pressure and healthy individuals and purifying the lipase enzyme utilizing gel filtration technology. Besides, the investigation studied the kinetics of the lipase enzyme, the impact of various substrate concentrations, the impact of pH and optimal temperature on the enzyme's activity, and the impact of reaction time on the enzyme's effectiveness. It was found that the effectiveness of the lipase enzyme reduced with raising the concentration of the synthesized compound, and the sort of inhibition was determined as competitive.

Investigating the Effect of Chemical Modifications on the Ribose Sugar Conformation, Watson-Crick Base Pairing, and Intrastrand Stacking Interactions: A Theoretical Approach.

Over the last few decades, chemically modified sugars have been incorporated into nucleic acid-based therapeutics to improve their pharmacological potential. Chemical modification can influence the sugar conformation, Watson-Crick hydrogen (W-C) bonding, and nucleobase stacking interactions, which play major roles in the structural integrity and dynamic properties of nucleic acid duplexes. In this study, we categorized 33 uridine (U*) and cytidine (C*) sugar modifications and calculated their sugar conformational parameters. We also calculated the Watson-Crick hydrogen bond energies of the modified RNA-type base pairs (U*:A and C*:G) using DFT and sSAPT0 methods. The W-C base pairing energy calculations suggested that the South-type modified sugar strengthens the C*:G base pair and weakens the U*:A base pair compared to the unmodified one. In contrast, the North-type sugar modifications form weaker C*:G base pair and marginally stronger U*:A base pair compared to the South-type modified sugars. Moreover, intrastrand base stacking energies were calculated for 15 modifications incorporated at the fourth position in 7-mer non-self-complementary RNA duplexes [(GCAU*GAC)2 and (GCAC*GAC)2], utilizing molecular dynamics simulation and quantum mechanical (DFT and sSAPT0) methods. The sugar modifications were found to have minimal effect on the intrastrand base-stacking interactions. However, the glycol nucleic acid modification disturbs the intrastrand base-stacking significantly, corroborating the experimental data.

Enzymatic Functionalization of RNA Oligonucleotides by Terminal Uridylyl Transferase Using Fluorescent and Clickable Nucleotide Analogs.

We report a systematic study on controlling the enzyme activity of a terminal uridylyl transferase (TUTase) called SpCID1, which provides methods to effect site-specific incorporation of a single modified nucleotide analog at the 3'-end of an RNA oligonucleotide (ON). Responsive heterocycle-modified fluorescent UTP probes that are useful in analyzing non-canonical nucleic acid structures and azide- and alkyne-modified UTP analogs that are compatible for chemoenzymatic functionalization were used as study systems. In the first strategy, we balanced the concentration of essential metal ion cofactors (Mg2+ and Mn2+ ions) to restrict the processivity of the enzyme, which gave a very good control on the incorporation of clickable nucleotide analogs. In the second approach, borate that complexes with 2' and 3' oxygen atoms of a ribose sugar was used as a reversibly binding chelator to block repeated addition of nucleotide analogs. Notably, in the presence of heterocycle-modified fluorescent UTPs, we obtained single-nucleotide incorporated RNA products in reasonable yields, while with clickable nucleotides yields were very good. Further, 3'-end azide- and alkyne-labeled RNA ONs were post-enzymatically functionalized by CuAAC and SPAAC reactions with fluorescent probes. These strategies broaden the scope of TUTase in site-specifically installing modifications of different types onto RNA for various applications.

Elucidation of inhibitory effects of bioactive anthraquinones towards formation of DNA advanced glycation end products (DNA-AGEs)

DNA is essential in biological processes as it directs transcription and translation assisting in RNA and protein synthesis. Extended periods of elevated blood glucose levels cause non-enzymatic DNA glycation, which results in the formation of DNA-AGEs and the production of free radicals, causing structural perturbation of DNA. In this work, we have investigated the glycation of calf thymus (ct-DNA) DNA and examined its inhibition by two anthraquinone derivatives, purpurin and aloin. Ribose sugar served as the glycating agent inducing non-enzymatic glycation of DNA and subsequent DNA-AGEs formation. UV–vis and fluorescence spectroscopic methods were utilized to characterize DNA-AGE formation in vitro. Circular dichroism (CD) spectroscopy was used to observe the structural disruption of DNA caused by glycation. The changes in AGEs fluorescence intensity and melting temperature (Tm) were measured to assess the inhibition of glycation process by aloin and purpurin. These derivatives demonstrated inhibitory effects via binding to glycating sites of ct-DNA or by scavenging free radicals generated during glycation. The current study elucidates the inhibitory actions of aloin and purpurin on DNA glycation, suggesting their possible applications in mitigating the adverse consequences linked to increased ribose concentrations.

Infrared Spectroscopy of RNA Nucleosides in a Wide Range of Temperatures.

The RNA world hypothesis suggests that early cellular ancestors relied solely on RNA molecules for both genetic information storage and cellular functions. RNA, composed of four nucleosides-adenosine, guanosine, cytidine, and uridine-forms the basis of this theory. These nucleosides consist of purine nucleobases, adenine and guanine, and pyrimidine nucleobases, cytosine and uracil, bonded to ribose sugar. Notably, carbonaceous chondrite meteorites have revealed the presence of these bases and sugar, hinting at the potential existence of nucleosides in space. This study aims to present the infrared spectra of four RNA nucleosides commonly found in terrestrial biochemistry, facilitating their detection in space, especially in astrobiological and astrochemical contexts. Laboratory measurements involved obtaining mid- and far-IR spectra at three temperatures (-180 °C, room temperature, and +180 °C), followed by calculating molar extinction coefficients (ε) and integrated molar absorptivities (ψ) for corresponding bands. These spectral data, along with ε and ψ values, serve to provide quantitative insights into the presence and relative abundance of nucleosides in space and aid in their detection.

The clinical potential of l-oligonucleotides: challenges and opportunities.

Chemically modified nucleotides are central to the development of biostable research tools and oligonucleotide therapeutics. In this context, l-oligonucleotides, the synthetic enantiomer of native d-nucleic acids, hold great promise. As enantiomers, l-oligonucleotides share the same physical and chemical properties as their native counterparts, yet their inverted l-(deoxy)ribose sugars afford them orthogonality towards the stereospecific environment of biology. Notably, l-oligonucleotides are highly resistant to degradation by cellular nucleases, providing them with superior biostability. As a result, l-oligonucleotides are being increasingly utilized for the development of diverse biomedical technologies, including molecular imaging tools, diagnostic biosensors, and aptamer-based therapeutics. Herein, we present recent such examples that highlight the clinical potential of l-oligonucleotides. Additionally, we provide our perspective on the remaining challenges and practical considerations currently associated with the use of l-oligonucleotides and explore potential solutions that will lead to the broader adoption of l-oligonucleotides in clinical applications.

RNA Editing in Cancer Progression.

Coding and noncoding RNA molecules play their roles in ensuring cell function and tissue homeostasis in an ordered and systematic fashion. RNA chemical modifications can occur both at bases and ribose sugar, and, similarly to DNA and histone modifications, can be written, erased, and recognized by the corresponding enzymes, thus modulating RNA activities and fine-tuning gene expression programs. RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification in normal physiological processes. By altering the sequences of mRNAs, it makes them different from the corresponding genomic template. Hence, edited mRNAs can produce protein isoforms that are functionally different from the corresponding genome-encoded variants. Abnormalities in regulatory enzymes and changes in RNA-modification patterns are closely associated with the occurrence and development of various human diseases, including cancer. To date, the roles played by RNA modifications in cancer are gathering increasing interest. In this review, we focus on the role of RNA editing in cancer transformation and provide a new perspective on its impact on tumorigenesis, by regulating cell proliferation, differentiation, invasion, migration, stemness, metabolism, and drug resistance.

Analysis on the in-silico ensemble methods for 3D modelling of ssDNA aptamers

Understanding the 3-D structure of nucleic acid aptamers is important for the rational design of aptamer-based constructs in various applications, including for developing aptasensors. Herein, a simple approach for 3D modelling of ssDNA aptamers through an ensemble of web applications has been described. The procedure utilized 30 aptamers whose 3D XRD or NMR experimental structures are available for validation. As a first step, the primary sequences of ssDNA aptamers were transformed into 2D structures using six widely used web applications: RNA fold, Vector builder, RNA Structure, UNA fold, Centroid fold, and IP Knot. The generated 2D structures were then passed through the RNA composer web application to generate 3D RNA structure, which in turn was converted to 3D DNA structures using various Visual Molecular Dynamics web applications that also include conversion of ribose sugar into deoxyribose sugar backbone and uracil to thiamine. The energy-minimized generated 3D structures were matched well with high accuracy to their experimental counterparts. This study identified that the Guanine residues are crucial in the aptamer 3D structure prediction and in algorithms that generate secondary structures. Further, the GC content (<50%), GC bond percentage (<60%), and G:C ratio (<1.12) act as limiting factors in predicting the 2D structures of aptamers. There were variations in the 2D structure predictions by the web applications, even though all these applications were a combination of the MFE, MEA, and McCaskill functions. Processing these structures through the web applications described above produced best-fit 3D structures with the experimental one, thus offering the present ensemble approach to reliably predict the 3D structure of aptamers for various applications.

Innate immune regulations and various siRNA modalities.

RNAi therapeutics are designed to produce the precise silencing effects against the gene-linked diseases which were known to be untreatable in the past. The highly immunostimulatory nature of siRNA enhances the off-target effects and easily get attacked by nucleases; hence, their modulation is essentially required for accurate alterations to be made in the structures to intensify the pharmacological attributes. The phosphonate modifications act as shield against undue phosphorylation effects, and the molecular changes in ribose sugar lowers the level of immunogenicity and increases the binding efficacy. When bases are substituted with virtual/or pseudo bases, they eventually reduce the off-target effects. These changes modulate the nucleic acid sensors and control the hyper-activation of innate immune response. Various modification designs based on STC (universal pattern), ESC, ESC + (advanced patterns) and disubstrate have been explored to silence the gene expression of various diseases e.g., hepatitis, HIV, influenza, RSV, CNV and acute kidney injury. This review describes the various innovative siRNA therapeutics and their implications on the developed immune regulations to silence the disease effects. siRNA causes the silencing effects through RISC processing. The innate immune signalling is induced by both TLR-dependent and TLR-independent pathways. Modification chemistries are utilized to modulate the immune response.

Integrated molecular and quantum mechanical approach to identify novel potent natural bioactive compound against 2′-O-methyltransferase (nsp16) of SARS-CoV-2

With the advent of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak, efforts are still in progress to find out a functional cure for the infection. Among the various protein targets, nsp16 capping protein is one of the vital targets for drug development as it protects the virus against the host cell nucleases and evading innate immunity. The nsp16 protein forms a heterodimer with a co-factor nsp10 and triggers 2′-O-methyltransferase activity which catalyzes the conversion of S-adenosyl methionine into S-adenosyl homocysteine. The free methyl group is transferred to the 2′-O position on ribose sugar at the 5′ end of mRNA to form the cap-1 structure which is essential for replication of the virus and evading the innate immunity of the host. In this study, we identify a potential lead natural bioactive compound against nsp16 protein by systematic cheminformatic analysis of more than 144k natural compounds. Virtual screening, molecular docking interactions, ADMET profiling, molecular dynamics (MD) simulations, molecular mechanics-generalized born surface area (MM-GBSA), free energy analysis and density functional theory analysis were used to discover the potential lead compound. Our investigation revealed that ZINC8952607 (methyl-[(6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)aminomethyl]BLAHone) has the greatest binding affinity and best pharmacokinetic parameters due to presence of carbazol and BLAHone (biaryl moiety). Further, time-dependent MD simulation analysis substantiates the stability and rigidness of nsp16 protein even after interaction with the lead compound. We believe that the compound ZINC8952607 might establish as a novel natural drug candidate against CoVID-19 infection. Communicated by Ramaswamy H. Sarma

Periodontal disease in chronic kidney disease patients: salivomics by Fourier-transform infrared spectroscopy.

It has been reported that 58% of individuals with chronic kidney disease (CKD) have moderate to advanced periodontitis due to alterations of pH and biochemical composition in the saliva. In fact, the composition of this important biofluid may be modulated by systemic disorders. Here we investigate the micro-reflectance Fourier-transform infrared spectroscopy (FTIR) spectra of saliva that CKD patients submitted to periodontal treatment, aiming to identify spectral biomarkers of kidney disease evolution and the effectiveness of periodontal treatment, proposing possible biomarkers of disease evolution. Saliva from 24 CKD patients-stage-5 men, 29 to 64years old-was evaluated in (i)patients starting periodontal treatment; (ii)patients 30days after periodontal treatment; and (iii)patients 90days after periodontal treatment. Our findings indicated that there are statistically relevant changes among the groups after 30 and 90days of periodontal treatment, when considering the overall spectra in the fingerprint region (800-1800cm-1). The key bands presenting good prediction power (area under the receiver operating characteristic curve >0.70) were related to poly (ADP-ribose) polymerase (PARP) conjugated to DNA at 883, 1031, and 1060cm-1 (carbohydrates at 1043 and 1049cm-1) and triglycerides (1461cm-1). Interestingly when analyzing the derivative spectra in the secondary structure region (1590-1700cm-1), we detected over-expression of the β-sheet class of secondary structures in 90days of periodontal treatment, possibly related to over-expression of human B-defensins. Conformational changes in ribose sugar in this region corroborate the interpretation concerning PARP detection. To our knowledge, PARP was detected for the first time in saliva samples of stage-5 CKD patients by FTIR. All observed changes were correctly interpreted in terms of intensive apoptosis and dyslipidemia due to kidney disease progression. Biomarkers due to CKD predominate in saliva, and the relative improvement in the periodontal state did not cause remarkable changes in the spectra of saliva.

Recycling of Polymerase Chain Reaction (PCR) Kits.

Polymerase chain reaction (PCR) kits have been used as common diagnosing tools during the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic, there was a shortage of PCR kits. So far, the ecosystem of a PCR kit is linear use; that is, kits are produced, used once, and disposed of as biolab waste. Here, we show that to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands, keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotide monophosphates that are then phosphorylated into triphosphates using a method that is developed from a recent publication. We used oligonucleotides with a 3'-terminal phosphorothioate (PS) backbone modification as nonhydrolyzable PCR primers, which are able to undergo the recycling process unchanged. The nuclease resistance of oligonucleotides with a ribose sugar modification was also evaluated, which showed worse recycling efficiency than PS-modified oligonucleotides. We successfully recycled both PCR primers and nucleotide monomers (∼75% yield). We demonstrate that the method allows for the direct reuse of PCR kits. We also show that the recycled primers can be isolated and then added to endpoint or quantitative PCR. This recycling approach provides a new path for circularly reusing nucleic acid materials in PCR kits.

TMAO Destabilizes RNA Secondary Structure via Direct Hydrogen Bond Interactions.

Trimethylamine N-oxide (TMAO) is an osmolyte that accumulates in cells in response to osmotic stress. TMAO stabilizes proteins by the entropic stabilization mechanism, which pictures TMAO as a nanocrowder that predominantly destabilizes the unfolded state. However, the mechanism of action of TMAO on RNA is much less understood. Here, we use all-atom molecular dynamics simulations to investigate how TMAO interacts with a 12-nt RNA hairpin with a high melting temperature, and an 8-nt RNA hairpin, which has a relatively fluid native basin in the absence of TMAO. The use of the two hairpins with different free energy of stabilization allows us to probe the origin of the destabilization effect of TMAO on RNA molecules without the possibility of forming tertiary interactions. We generated multiple trajectories using all-atom molecular dynamics (MD) simulations in explicit water by employing AMBER and CHARMM force fields, both in the absence and presence of TMAO. We observed qualitatively similar RNA-TMAO interaction profiles from the simulations using the two force fields. TMAO hydrogen bond interactions are largely depleted around the paired RNA bases and ribose sugars. In contrast, we show that the oxygen atom in TMAO, the hydrogen bond acceptor, preferentially interacts with the hydrogen bond donors in the solvent exposed bases, such as those in the stem-loop and the destabilized base stacks in the unfolded state, especially in the marginally stable 8-nt RNA hairpin. The predicted destabilization mechanism through TMAO-RNA hydrogen bond interactions could be tested using two-dimensional IR spectroscopy. Since TMAO does not significantly interact with the hydroxyl group of the ribose sugars, we predict that similar results must also hold for DNA.

Prediction and Motif Analysis of 2’-O-methylation Using a Hybrid Deep Learning Model from RNA Primary Sequence and Nanopore Signals

Background: 2’-O-Methylation (2’-O-Me) is a post-transcriptional RNA modification that occurs in the ribose sugar moiety of all four nucleotides and is abundant in both coding and non-coding RNAs. Accurate prediction of each subtype of 2’-O-Me (Am, Cm, Gm, Um) helps understand their role in RNA metabolism and function. Objective: This study aims to build models that can predict each subtype of 2’-O-Me from RNA sequence and nanopore signals and exploit the model interpretability for sequence motif mining. Methods: We first propose a novel deep learning model DeepNm to better capture the sequence features of each subtype with a multi-scale framework. Based on DeepNm, we continue to propose HybridNm, which combines sequences and nanopore signals through a dual-path framework. The nanopore signalderived features are first passed through a convolutional layer and then merged with sequence features extracted from different scales for final classification. Results: A 5-fold cross-validation process on Nm-seq data shows that DeepNm outperforms two stateof- the-art 2’-O-Me predictors. After incorporating nanopore signal-derived features, HybridNm further achieved significant improvements. Through model interpretation, we identified not only subtypespecific motifs but also revealed shared motifs between subtypes. In addition, Cm, Gm, and Um shared motifs with the well-studied m6A RNA methylation, suggesting a potential interplay among different RNA modifications and the complex nature of epitranscriptome regulation. Conclusion: The proposed frameworks can be useful tools to predict 2’-O-Me subtypes accurately and reveal specific sequence patterns.

Metabolic flexibilities and vulnerabilities in the pentose phosphate pathway of the zoonotic pathogen Toxoplasma gondii.

Metabolic pathways underpin the growth and virulence of intracellular parasites and are therefore promising antiparasitic targets. The pentose phosphate pathway (PPP) is vital in most organisms, providing a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose sugar for nucleotide synthesis; however, it has not yet been studied in Toxoplasma gondii, a widespread intracellular pathogen and a model protozoan organism. Herein, we show that T. gondii has a functional PPP distributed in the cytoplasm and nucleus of its acutely-infectious tachyzoite stage. We produced eight parasite mutants disrupting seven enzymes of the PPP in T. gondii. Our data show that of the seven PPP proteins, the two glucose-6-phosphate dehydrogenases (TgG6PDH1, TgG6PDH2), one of the two 6-phosphogluconate dehydrogenases (Tg6PGDH1), ribulose-5-phosphate epimerase (TgRuPE) and transaldolase (TgTAL) are dispensable in vitro as well as in vivo, disclosing substantial metabolic plasticity in T. gondii. Among these, TgG6PDH2 plays a vital role in defense against oxidative stress by the pathogen. Further, we show that Tg6PGDH2 and ribulose-5-phosphate isomerase (TgRPI) are critical for tachyzoite growth. The depletion of TgRPI impairs the flux of glucose in central carbon pathways, and causes decreased expression of ribosomal, microneme and rhoptry proteins. In summary, our results demonstrate the physiological need of the PPP in T. gondii while unraveling metabolic flexibility and antiparasitic targets.

Structural Significance of Conformational Preferences and Ribose-Ring-Puckering of Hyper Modified Nucleotide 5'-Monophosphate 2-Methylthio Cyclic N6-Threonylcarbamoyladenosine (p-ms2ct6A) Present at 37th Position in Anticodon Loop of tRNALys.

Structural significance of conformational preferences and ribose ring puckering of newly discovered hyper modified nucleotide, 5'-monophosphate 2-methylthio cyclic N6-threonylcarbamoyladenosine (p-ms2ct6A) have been investigated using quantum chemical semi-empirical RM1 and molecular dynamics simulation techniques. Automated geometry optimization of most stable structure of p-ms2ct6A has also been carried out with the help of abinitio (HF SCF, DFT) as well as semi empirical quantum chemical (RM1, AM1, PM3, and PM6) methods. Most stable structure of p-ms2ct6A is stabilized by intramolecular interactions between N(3)…HC(2'), N(1)…HC(16), O(13)…HC(15), and O(13)…HO(14). The torsion angles alpha (α) and beta (β) show the significant characteristic patterns with the involvement of intramolecular hydrogen bonding to provide stability to the p-ms2ct6A. Further, molecular dynamics simulations of p-ms2ct6A revealed the role of ribose sugar ring puckering i.e. C2'-endo and C3'-endo on the structural dynamics of ms2ct6A side chain. The modified nucleotide p-ms2ct6A periodically prefers both the C2'-endo and C3'-endo sugar with 'anti' and 'syn' conformations. This property of p-ms2ct6A could be useful to recognize the starting ANN codons. All atom explicit MD simulation of anticodon loop (ACL) of tRNALys of Bacillus subtilis containing ms2ct6A at 37th position showed the U-turn feature, base stacking ability with other adjacent bases and hydrogen bonding interactions similar to the isolated base p-ms2ct6A. The ribose sugar puckering contributes to the orientation of the side chain conformation of p-ms2ct6A. Thus, the present study could be helpful to understand the structure-function relationship of the hypermodified nucleoside, ms2ct6A in recognition of the proper codons AAA/AAG during protein biosynthesis.

Harnessing Nature's Molecular Recognition Capabilities to Map and Study RNA Modifications.

RNA editing or "epitranscriptomic modification" refers to the processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid. These chemical alterations can be found on either the ribose sugar or the nucleobase, and although many are "silent" and do not change the Watson-Crick-Franklin code of the RNA, others result in recoding events. More than 170 RNA modifications have been identified so far, each having a specific biological purpose. Additionally, dysregulated RNA editing has been linked to several types of diseases and disorders. As new modifications are discovered and our understanding of their functional impact grows, so does the need for selective methods of identifying and mapping editing sites in the transcriptome.The most common methods for studying RNA modifications rely on antibodies as affinity reagents; however, antibodies can be difficult to generate and often have undesirable off-target binding. More recently, selective chemical labeling has advanced the field by offering techniques that can be used for the detection, enrichment, and quantification of RNA modifications. In our method using acrylamide for inosine labeling, we demonstrated the versatility with which this approach enables pull-down or downstream functionalization with other tags or affinity handles. Although this method did enable the quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely because of the similar reactivity profiles of inosine and pseudouridine or other nucleobases.Seeking to overcome the inherent limitations of antibodies and chemical labeling methods, a more recent approach to studying the epitranscriptome is through the repurposing of proteins and enzymes that recognize modified RNA. Our laboratory has used Endonuclease V, a repair enzyme that cleaves inosine-containing RNAs, and reprogrammed it to instead bind inosine. We first harnessed EndoV to develop a preparative technique for RNA sequencing that we termed EndoVIPER-seq. This method uses EndoV to enrich inosine-edited RNAs, providing better coverage in RNA sequencing and leading to the discovery of previously undetected A-to-I editing sites. We also leveraged EndoV to create a plate-based immunoassay (EndoVLISA) to quantify inosine in cellular RNA. This approach can detect differential A-to-I editing levels across tissue types or disease states while being independent of RNA sequencing, making it cost-effective and high-throughput. By harnessing the molecular recognition capabilities of this enzyme, we show that EndoV can be repurposed as an "anti-inosine antibody" to develop new methods of detecting and enriching inosine from cellular RNA.Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications, and exploiting the affinity of these biomolecules offers a promising new direction for the field of epitranscriptomics.

Infrared spectroscopy and flow cytometry studies on the apoptotic effect of nano-chrysin in HeLa cells

Mapping the structural changes in membrane lipids, proteins, polysaccharides and nucleic acids has opened new channels for understanding the mode of action of anticancer natural products. Earlier, we synthesized chrysin nanoparticles (NChr) with good bioavailability, and characterized its size, surface charge, entrapment efficiency, and drug release pattern using PLGA polymer. NChr induced concentration dependent cytotoxicity in HeLa cells with an IC50 of 61.54 ± 1.2 µM in comparison with free chrysin with IC50 of 86.51 ± 2.9 µM. Since nanoparticles interact dynamically with cell membranes, organelles, proteins and DNA, it is necessary to understand the interplay of nanodrug induced macromolecular changes in cancer cells. In this work, we obtained signatures of NChr-induced biochemical changes in HeLa cells by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy technique coupled with flow cytometry. NChr induced cell membrane disruption, G1 phase cell cycle arrest, and increased externalization of phosphatidylserine leading to apoptosis indicating the biochemical perturbations in membrane lipids and DNA of HeLa cells in comparison with untreated cells. The 1300–1000 cm−1 spectral region indicated NChr interaction with the ribose sugar backbone and DNA denaturation. Spectral range 1800–1400 cm−1 indicated a concentration dependent decrease in α helical and β sheet structures which may lead to protein degradation during apoptosis. The spectral range 3000–2800 cm−1 indicated the lipid peroxidation in response to NChr treatment. This is the first study describing the bio-macromolecular changes induced by a nano encapsulated drug and can pave the way to investigate unconventional modes of action for bioactive formulations.

The Inhibitory Potential of 2'-dihalo Ribonucleotides against HCV: Molecular Docking, Molecular Simulations, MM-BPSA, and DFT Studies.

Sofosbuvir is the first approved direct-acting antiviral (DAA) agent that inhibits the HCV NS5B polymerase, resulting in chain termination. The molecular models of the 2′-dihalo ribonucleotides used were based on experimental biological studies of HCV polymerase inhibitors. They were modeled within HCV GT1a and GT1b to understand the structure–activity relationship (SAR) and the binding interaction of the halogen atoms at the active site of NS5B polymerase using different computational approaches. The outputs of the molecular docking studies indicated the correct binding mode of the tested compounds against the active sites in target receptors, exhibiting good binding free energies. Interestingly, the change in the substitution at the ribose sugar was found to produce a mild effect on the binding mode. In detail, increasing the hydrophobicity of the substituted moieties resulted in a better binding affinity. Furthermore, in silico ADMET investigation implied the general drug likeness of the examined derivatives. Specifically, good oral absorptions, no BBB penetration, and no CYP4502D6 inhibitions were expected. Likely, the in silico toxicity studies against several animal models showed no carcinogenicity and high predicted TD50 values. The DFT studies exhibited a bioisosteric effect between the substituents at the 2′-position and the possible steric clash between 2′-substituted nucleoside analogs and the active site in the target enzyme. Finally, compound 6 was subjected to several molecular dynamics (MD) simulations and MM-PBSA studies to examine the protein-ligand dynamic and energetic stability.

Structure‐activity relationships for several series of fragment‐based inhibitors that target Trichomonas vaginalis nucleoside ribohydrolase enzymes

Trichomoniasis, the most common sexually transmitted infection in the world, is caused by the parasitic protozoan Trichomonas vaginalis. Upon infection, T. vaginalis has been known to cause a variety of adverse effects as well as predisposition to other illnesses. Trichomoniasis is commonly treated with 5‐nitroimidazole drugs; however, some strains have developed resistance to these drugs and novel treatments are needed. Since T. vaginalis cannot synthesize nucleobases de novo, it relies on nucleoside ribohydrolases within its pyrimidine and purine salvage pathways in order to acquire nucleobases from its host. Nucleoside ribohydrolases, such as adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH), cleave the N‐glycosidic bond of purine and pyrimidine nucleosides resulting in the formation of a nucleobase and a ribose sugar. Inhibiting the AGNH or UNH enzymes would block the salvage pathway resulting in parasite cell death. Fragment screening of AGNH and UNH identified ligand‐efficient scaffolds to serve as medicinal chemistry starting points for drug design. A variety of phenyl pyridine, phenyl pyrimidine, and phenyl pyrazine compounds were then synthesized and screened for inhibitory properties. 1H and 19F NMR‐based activity assays were employed, using adenosine and 5‐fluorouridine as substrates for AGNH and UNH, respectively. It was found that compounds containing a hydroxyl group at the 2‐position resulted in lower IC50 values for AGNH, and that the UNH IC50 was lower for compounds with hydroxy or methoxy functional groups at the 2’ and 3’ positions. With the enzyme inhibition data at hand structure‐activity relationships are being derived and will be used in combination with molecular modeling to design the next generation of compounds to be synthesized. Structural data is also being pursued for both enzymes to guide compound design. Highly purified AGNH and UNH, including selenomethionine‐AGNH, have been prepared for crystallography collaborations.