Sod1-deficient cells are impaired in formation of the modified nucleosides mcm5s2U and yW in tRNA.

Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm5), 5-methoxycarbonylmethyl (mcm5), or 5-methoxycarbonylhydroxymethyl (mchm5) side-chain. The presence of these side-chains allows proper pairing with cognate codons and they are particularly important in tRNA species where the U34 residue is also modified with a 2-thio (s2) group. The first step in synthesis of the ncm5, mcm5, and mchm5 side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1, which encodes the cytosolic Cu,Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-modified U34 residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNAPhe Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells.

1mΨ influences the performance of various positive-stranded RNA virus-based replicons.

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.

Analysis of RNA and Its Modifications.

Ribonucleic acids (RNAs) are key biomolecules responsible for the transmission of genetic information, the synthesis of proteins, and modulation of many biochemical processes. They are also often the key components of viruses. Synthetic RNAs or oligoribonucleotides are becoming more widely used as therapeutics. In many cases, RNAs will be chemically modified, either naturally via enzymatic systems within a cell or intentionally during their synthesis. Analytical methods to detect, sequence, identify, and quantify RNA and its modifications have demands that far exceed requirements found in the DNA realm. Two complementary platforms have demonstrated their value and utility for the characterization of RNA and its modifications: mass spectrometry and next-generation sequencing. This review highlights recent advances in both platforms, examines their relative strengths and weaknesses, and explores some alternative approaches that lie at the horizon.

The Bacillus subtilis ywbD gene encodes RlmQ, the 23S rRNA methyltransferase forming m7G2574 in the A-site of the peptidyl transferase center.

Ribosomal RNA contains many posttranscriptionally modified nucleosides, particularly in the functional parts of the ribosome. The distribution of these modifications varies from one organism to another. In Bacillus subtilis, the model organism for Gram-positive bacteria, mass spectrometry experiments revealed the presence of 7-methylguanosine (m7G) at position 2574 of the 23S rRNA, which lies in the A-site of the peptidyl transferase center of the large ribosomal subunit. Testing several m7G methyltransferase candidates allowed us to identify the RlmQ enzyme, encoded by the ywbD open reading frame, as the MTase responsible for this modification. The enzyme methylates free RNA and not ribosomal 50S or 70S particles, suggesting that modification occurs in the early steps of ribosome biogenesis.

Exploring urinary modified nucleosides as biomarkers for diabetic retinopathy: Development and validation of a ultra performance liquid chromatography-tandem mass spectrometry method.

The dynamic modification of RNA plays a crucial role in biological regulation and is strongly linked to human disease development and progression. Notably, modified nucleosides in urine have shown promising potential as early diagnostic biomarkers for various conditions. In this study, we developed and validated a rapid, sensitive, and accurate UPLC-MS/MS method for quantifying eight types of modified nucleosides (N1-methyladenosine (m1A), N6-methyladenosine (m6A), 5-methyluridine (m5U), 5-taurinomethyl-2-thiouridine (τm5s2U), 5-methylcytidine (m5C), 2'-O-methylcytidine (Cm), N1-methylguanosine (m1G), and N7-methylguanosine (m7G) in human urine. Using the method, we measured the urinary concentrations of m1A, m6A, m5U, τm5s2U, m5C, Cm, m1G, and m7G in a total of 21 control individuals and 23 patients diagnosed with diabetic retinopathy (DR). Cm levels showed promise as a diagnostic marker for diabetic retinopathy (DR), with a significant value (P < 0.01) and an AUC of 0.735. Other modified nucleosides also exhibited significant differences within specific subpopulations. As non-proliferative diabetic retinopathy (NPDR) signifies the latent early stage of diabetic retinopathy, we developed a multivariate linear model that integrates patients' sex, age, height, and urinary concentration of modified nucleosides which aims to predict and differentiate between healthy individuals, NPDR patients, and proliferative diabetic retinopathy (PDR) patients. Encouragingly, the model achieved satisfactory accuracy rates: healthy (81%), NPDR (75%), and PDR (80%). Our findings provide valuable insights into the development of an early, cost-effective, and noninvasive diagnostic approach for diabetic retinopathy.

Characterizing Benzo[a]pyrene Adducts in Transfer RNAs Using Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS).

The activated forms of the environmental pollutant benzo[a]pyrene (B[a]P), such as benzo[a]pyrene diol epoxide (BPDE), are known to cause damage to genomic DNA and proteins. However, the impact of BPDE on ribonucleic acid (RNA) remains unclear. To understand the full spectrum of potential BPDE-RNA adducts formed, we reacted ribonucleoside standards with BPDE and characterized the reaction products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To understand the potential types of adducts that could form with biological RNAs, eukaryotic transfer RNAs (tRNAs) were also reacted with BPDE. The isolation and analysis of the modified and adducted ribonucleosides using LC-MS/MS revealed several BPDE derivatives of post-transcriptional modifications. The approach outlined in this work enables the identification of RNA adducts from BPDE, which can pave the way for understanding the potential impacts of such adducts on the higher-order structure and function of modified RNAs.

The mRNA Vaccine Revolution: COVID-19 Has Launched the Future of Vaccinology.

During the COVID-19 pandemic, mRNA (mRNA) vaccines emerged as leading vaccine candidates in a record time. Nonreplicating mRNA (NRM) and self-amplifying mRNA (SAM) technologies have been developed into high-performing and clinically viable vaccines against a range of infectious agents, notably SARS-CoV-2. mRNA vaccines demonstrate efficient in vivo delivery, long-lasting stability, and nonexistent risk of infection. The stability and translational efficiency of in vitro transcription (IVT)-mRNA can be further increased by modulating its structural elements. In this review, we present a comprehensive overview of the recent advances, key applications, and future challenges in the field of mRNA-based vaccinology.

Targeted quantitative metabolomics with a linear mixed-effect model for analysis of urinary nucleosides and deoxynucleosides from bladder cancer patients before and after tumor resection.

In the present study, we developed and validated a fast, simple, and sensitive quantitative method for the simultaneous determination of eleven nucleosides and deoxynucleosides from urine samples. The analyses were performed with the use of liquid chromatography coupled with triple quadrupole mass spectrometry. The sample pretreatment procedure was limited to centrifugation, vortex mixing of urine samples with a methanol/water solution (1:1, v/v), evaporation and dissolution steps. The analysis lasted 20 min and was performed in dynamic multiple reaction monitoring mode (dMRM) in positive polarity. Process validation was conducted to determine the linearity, precision, accuracy, limit of quantification, stability, recovery and matrix effect. All validation procedures were carried out in accordance with current FDA and EMA regulations. The validated method was applied for the analysis of 133 urine samples derived from bladder cancer patients before tumor resection and 24 h, 2 weeks, and 3, 6, 9, and 12 months after the surgery. The obtained data sets were analyzed using a linear mixed-effect model. The analysis revealed that concentration level of 2-methylthioadenosine was decreased, while for inosine, it was increased 24 h after tumor resection in comparison to the preoperative state. The presented quantitative longitudinal study of urine nucleosides and deoxynucleosides before and up to 12 months after bladder tumor resection brings additional prospective insight into the metabolite excretion pattern in bladder cancer disease. Moreover, incurred sample reanalysis was performed proving the robustness and repeatability of the developed targeted method.

Integrated Metabolomic and Transcriptomic Analysis of Modified Nucleosides for Biomarker Discovery in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers. The biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is the truncal driver mutation of most cases of ccRCC. Cancer cells are metabolically reprogrammed and excrete modified nucleosides in larger amounts due to their increased RNA turnover. Modified nucleosides occur in RNAs and cannot be recycled by salvage pathways. Their potential as biomarkers has been demonstrated for breast or pancreatic cancer. To assess their suitability as biomarkers in ccRCC, we used an established murine ccRCC model, harboring Vhl, Trp53 and Rb1 (VPR) knockouts. Cell culture media of this ccRCC model and primary murine proximal tubular epithelial cells (PECs) were investigated by HPLC coupled to triple-quadrupole mass spectrometry using multiple-reaction monitoring. VPR cell lines were significantly distinguishable from PEC cell lines and excreted higher amounts of modified nucleosides such as pseudouridine, 5-methylcytidine or 2'-O-methylcytidine. The method's reliability was confirmed in serum-starved VPR cells. RNA-sequencing revealed the upregulation of specific enzymes responsible for the formation of those modified nucleosides in the ccRCC model. These enzymes included Nsun2, Nsun5, Pus1, Pus7, Naf1 and Fbl. In this study, we identified potential biomarkers for ccRCC for validation in clinical trials.

Nucleoside Derivatives of 2,6-Diaminopurine Antivirals: Base-Modified Nucleosides with Broad-Spectrum Antimicrobial Properties.

The plethora of viral outbreaks experienced in the last decade, together with the widespread distribution of many re-emerging and newly emerging viruses, emphasize the urgent need for novel broad-spectrum antivirals as tools for early intervention in case of future epidemics. Non-natural nucleosides have been at the forefront for the treatment of infectious diseases for many years and still represent one of the most successful classes of antiviral molecules on the market. In the attempt to explore the biologically relevant chemical space of this class of antimicrobials, we describe herein the development of novel base-modified nucleosides by converting previously identified 2,6-diaminopurine antivirals into the corresponding D/L ribonucleosides, acyclic nucleosides and prodrug derivatives. A phenotypic screening against viruses belonging to different families (Flaviviridae, Coronaviridae, Retroviridae) and against a panel of Gram-positive and Gram-negative bacteria, allowed to identify a few interesting molecules with broad-spectrum antimicrobial activities.

Inherently Emissive Puromycin Analogues for Live Cell Labelling.

Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me2 N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow-up chemistry. The 3,3-difluoroazetidine-containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons.

Synthesis of 6-Alkynylated Purine-Containing DNA via On-Column Sonogashira Coupling and Investigation of Their Base-Pairing Properties.

Synthetic unnatural base pairs have been proven to be attractive tools for the development of DNA-based biotechnology. Our group has very recently reported on alkynylated purine-pyridazine pairs, which exhibit selective and stable base-pairing via hydrogen bond formation between pseudo-nucleobases in the major groove of duplex DNA. In this study, we attempted to develop an on-column synthesis methodology of oligodeoxynucleotides (ODNs) containing alkynylated purine derivatives to systematically explore the relationship between the structure and the corresponding base-pairing ability. Through Sonogashira coupling of the ethynyl pseudo-nucleobases and CPG-bound ODNs containing 6-iodopurine, we have demonstrated the synthesis of the ODNs containing three NPu derivatives (NPu1, NPu2, NPu3) as well as three OPu derivatives (OPu1, OPu2, OPu3). The base-pairing properties of each alkynylated purine derivative revealed that the structures of pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that OPu1 bearing 2-pyrimidinone exhibits higher stability to the complementary NPz than the original OPu, thereby demonstrating the potential of the on-column strategy for convenient screening of the alkynylated purine derivatives with superior pairing ability.

Base pairs with 4-amino-3-nitrobenzonitrile: comparison with the natural WC pairs. Dimer and tetramer forms, Infrared and Raman spectra, and several proposed antiviral modified nucleosides.

Base pairs of 4-amino-3-nitrobenzonitrile (4A-3NBN) molecule with uracil, thymine and cytosine nucleobases were optimized and compared to natural Watson-Crick (WC) pairs. The slightly greater flexibility of the -NO2 group of 4A-3NBN than the N3-H group of the natural nucleobases together with a noticeable higher dipole moment of its pairs can facilitate disruption of the DNA/RNA helix formation. Several new mutagenic modified nucleosides with 4A-3NBN and 3-amino-2-nitrobenzonitrile (3A-2NBN) were proposed as antiviral prodrugs and their base pairs optimized. The special characteristics of these prodrugs appear appropriated for their clinical use. The counterpoise (CP) corrected interaction energies of the base pairs were calculated and compared to the natural ones. The M06-2X DFT method was used for this purpose. The molecular structure of 4A-3NBN was analyzed in detail and the crystal unit cell was simulated by a tetramer form and eight dimer forms. The performance of the B3LYP, X3LYP and M06-2X methods was tested on the vibrational wavenumbers in the monomer, dimer and tetramer forms of 4A-3NBN. The observed IR and Raman bands were assigned according to the optimum dimer II form determined by B3LYP and by the tetramer form calculated by M06-2X, which is the expected unit cell that forms the crystal net. The two best scaling procedures were used.Communicated by Ramaswamy H. Sarma.

Metabolic Analysis of Nucleosides/Bases in the Urine and Serum of Patients with Alcohol-Associated Liver Disease.

Accumulating evidence supports the important role of RNA modifications in liver disease pathogenesis. However, RNA modifications in alcohol-associated liver disease (ALD) have not yet been reported. Modified ribonucleosides/bases are products of RNA degradation; therefore, we investigated whether modified ribonucleosides/bases in human urine and serum are changed and whether these changes are associated with the severity of ALD. Human urine and serum samples from patients with ALD and appropriate controls were collected. Free nucleosides/bases were extracted from these samples and quantified using untargeted and targeted metabolomic approaches. Thirty-nine and forty free nucleosides/bases were respectively detected in human urine and serum samples. Twelve and eleven modified nucleosides are significantly changed in patients' urine and serum (q < 0.05 and fold-change > 20%). The abundance of modified nucleobase and ribonucleoside, 7,9-dimethylguanine in urine and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in serum are strongly associated with the severity of ALD. Spearman's rank correlation coefficient of these two metabolites with the Model for End-stage Liver Disease (MELD) score are 0.66 and 0.74, respectively. Notably, the abundance changes in these two metabolites are sufficiently large to distinguish severe alcohol-associate hepatitis (AH) from non-severe ALD and non-severe ALD from healthy controls.

Ultrasensitive Simultaneous Detection of Multiple Rare Modified Nucleosides as Promising Biomarkers in Low-Put Breast Cancer DNA Samples for Clinical Multi-Dimensional Diagnosis.

Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 µg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.

Synthesis of 4'-C-(Aminoethyl)thymidine and 4'-C-[(N-Methyl)aminoethyl] Thymidine Nucleosides to Enhance DNA Stability.

Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N3 -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20).

t6A and ms2t6A Modified Nucleosides in Serum and Urine as Strong Candidate Biomarkers of COVID-19 Infection and Severity.

SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.

Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs.

The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2'-deoxy-2'-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%-88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6-1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

Radiosensitization of PC3 Prostate Cancer Cells by 5-Thiocyanato-2'-deoxyuridine.

PURPOSE: The radiosensitizing properties of uracil analogs modified in the C5 position are very interesting in the context of their effectiveness and safety in radiation therapy. Recently, radiation chemical studies have confirmed that 5-thiocyanato-2'-deoxyuridine (SCNdU) undergoes dissociation induced by an excess electron attachment and established this nucleoside as a potential radiosensitizer. In this paper, we verify the sensitizing properties of SCNdU at the cellular level and prove that it can effectively enhance ionizing radiation-induced cellular death. METHODS AND MATERIALS: Prostate cancer cells were treated with SCNdU and irradiated with X rays. The cytotoxicity of SCNdU was determined by MTT test. Cell proliferation was assessed using a clonogenic assay. Cell cycle analyses, DNA damage, and cell death analyses were performed by flow cytometry. RESULTS: SCNdU treatment significantly suppressed the proliferation and increased the radiosensitivity of prostate cancer cells. The radiosensitizing effect expressed by the dose enhancement factor is equal to 1.69. Simultaneous exposure of cells to SCNdU and radiation causes an increase in the fraction of the most radiosensitive G2/M phase, enhancement of the histone H2A.X phosphorylation level, and apoptosis induction. Finally, SCNdU turned out to be marginally cytotoxic in the absence of ionizing radiation. CONCLUSIONS: Our findings indicate that SCNdU treatment enhances the radiosensitivity of prostate cancer cells in a manner associated with the cell cycle regulation, double strand formation, and a slight induction of apoptosis.