Protonation-Dependent Sequencing of 5-Formylcytidine in RNA

Chemical modification of cytidine in non-coding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remains unknown due to a lack of sensitive site-specific sequencing technologies. Here we report a protonation-dependent sequencing reaction for detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human tRNA. Finally, we demonstrate the utility of our method to characterize a patient-derived model of 5fC-deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA, and provide a molecular foundation for applying optimized sequencing reactions to better understand the role of oxidized RNA cytidine nucleobases in disease.

Effects of 5′,8′-Cyclo-2′-Deoxypurines on the Base ExcisionRepair of Clustered DNA Lesions in Nuclear Extracts of the XPC Cell Line

Clustered DNA lesions (CDL) containing 5′,8-cyclo-2′-deoxypurines (cdPus) are an example of extensive abnormalities occurring in the DNA helix and may impede cellular repair processes. The changes in the efficiency of nuclear base excision repair (BER) were investigated using (a) two cell lines, one of the normal skin fibroblasts as a reference (BJ) and the second from Xeroderma pigmentosum patients’ skin (XPC), and (b) synthetic oligonucleotides with single- and double-stranded CDL (containing 5′,8-cyclo-2′-deoxyadenosine (cdA) and the abasic (AP) site at various distances between lesions). The nuclear BER has been observed and the effect of both cdA isomers (5′R and 5′S) presence in the DNA was tested. CdPus affected the repair of the second lesion within the CDL. The BER system more efficiently processed damage in the vicinity of the ScdA isomer and changes located in the 3′-end direction for dsCDL and in the 5′-end direction for ssCDL. The presented study is the very first investigation of the repair processes of the CDL containing cdPu considering cells derived from a Xeroderma pigmentosum patient.

Application of TaqMan Real-Time PCR for Detecting ‘Candidatus Arsenophonus Phytopathogenicus’ Infection in Sugar Beet

The γ-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ is assigned as the major pathogen of “Syndrome des basses richesses”, a sugar beet disease characterised by a reduction in the sugar content of taproots and biomass yield. Despite the economic impact of this bacteriosis, diagnostics for this important pathogen currently rely on end-point PCR detection. Herein, we introduce a TaqMan qPCR for diagnostics of the agent targeting genes encoding a heat shock protein of the Hsp20 family and mannose-6-phosphate isomerase. Quantitation with synthetic oligonucleotides as standard showed that the developed TaqMan qPCR assays enable the detection of up to 100 target copies. A comparison between the TaqMan qPCR and end-point PCR for ‘Ca. A. phytopathogenicus’ detection was carried out on 78 sugar beet samples from different locations in southern Germany. The newly developed assays enable the fast, reliable and sensitive detection of ‘Ca. A. phytopathogenicus’ in sugar beet.

Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.

When UDG and hAPE1 Meet Cyclopurines. How (5′R) and (5′S) 5′,8-Cyclo-2′-deoxyadenosine and 5′,8-Cyclo-2′-deoxyguanosine Affect UDG and hAPE1 Activity?

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.

Mutations in two neuroblastoma rat sarcoma oncogenes are associated with progression of haematologic malignancies in Nigeria

Although mutation in the RAS genes has become important in the evaluation of haematologic malignancies worldwide, developing countries like Nigeria are yet to integrate it as a diagnostic tool and prognostic indicator for improved therapy. This study determined mutations in codons 12 and 13 of NRAS gene in blood donors and haematologic malignant individuals using multiplex (AS-PCR) and Sanger sequencing, thus highlighting the mutations as helpful diagnostic and prognostic tool. AS-PCR was used to selectively amplify mutation-specific synthetic oligonucleotides from the cfDNA of 100 study participants (50 cancer patients and 50 blood donors). Percentage mutation of 31.0% was seen in NRAS_G12D gene while NRAS_G13C had 17.0%. Twenty nine (29.0%) of the NRAS_G12D mutations were found in haematopoietic malignant patients and 2.0% were found in blood donors, while 15.0% of the NRAS_G13C were found in the malignant patients, confirming the occurrence of NRAS gene mutations in haematologic cancers and predominance of the G-A transition. The highest rate of mutation was observed in leukaemia patients, having a significant association with codon 13 (p = 0.042). Stages 3 and 2 cancers each had the highest mutation rates of NRAS_G12D and NRAS_G13C, revealing possible link between these mutations and susceptibility and progression of haematologic malignancies, which is higher in leukaemia. Further NRAS mutation studies and its role in other cancers are advocated, especially targeted towards ameliorating diagnosis and prognostic therapy. Challenges related to diagnosis and management of haematologic cancer continue to persist in developing countries like Nigeria. Thus, there is a need to go beyond studying the incidence and distribution pattern of these malignancies to capturing immunogenetic parameters of affected individuals.

Hydrogel based lipid-oligonucleotides: a new route to self-delivery of therapeutic sequences

Synthetic OligoNucleotides (ON) provide promising therapeutic tools for controlling specifically genetic expression in a broad range of diseases from cancers to viral infections. Beside their chemical stability and intracellular delivery,...

Evaluation of GENECUBE Mycoplasma for the detection of macrolide-resistant Mycoplasma pneumoniae

Introduction. Resistance against macrolide antibiotics in Mycoplasma pneumoniae is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have attractive features for the identification of Mycoplasma pneumoniae as well as its resistance-associated mutations of 23S ribosomal RNA (rRNA). Hypothesis/Gap Statement. The automated molecular diagnostic sytem can identify macrolide-resistant M. pneumoniae . Aim. To assess the performance of an automated molecular diagnostic system, GENECUBE Mycoplasma, in the detection of macrolide resistance-associated mutations. Methodology. To evaluate whether the system can distinguish mutant from wild-type 23S rRNA, synthetic oligonucleotides mimicking known mutations (high-level macrolide resistance, mutation in positions 2063 and 2064; low-level macrolide resistance, mutation in position 2067) were assayed. To evaluate clinical oropharyngeal samples, purified nucleic acids were obtained from M. pneumoniae -positive samples by using the GENECUBE system from nine hospitals. After confirmation by re-evaluation of M. pneumoniae positivity, Sanger-based sequencing of 23S rRNA and mutant typing using GENECUBE Mycoplasma were performed. Results. The system reproducibly identified all synthetic oligonucleotides associated with high-level macrolide resistance. Detection errors were only observed for A2067G (in 2 of the 10 measurements). The point mutation in 23S rRNA was detected in 67 (26.9 %) of 249 confirmed M. pneumoniae -positive clinical samples. The mutations at positions 2063, 2064 and 2617 were observed in 65 (97.0 %), 2 (3.0 %) and 0 (0.0 %) of the 67 samples, respectively. The mutations at positions 2063 and 2064 were A2063G and A2064G, respectively. The results from mutant typing using GENECUBE Mycoplasma were in full agreement with the results from sequence-based typing. Conclusion. GENECUBE Mycoplasma is a reliable test for the identification of clinically significant macrolide-resistant M. pneumoniae .

Assessment of the therapeutic potential of anti-miR 24 and anti-miR 34 in cardiac diseases

Purpose: To study the therapeutic effects of anti-miR-24 and anti-miR-34 in cardiac diseasesMethods: H9c2 rat cardiomyocyte cell lines were transfected with the synthetic oligonucleotides antimiR-24 and anti-miR-34 which reduced the expressions of miR-24 and miR-34. Proliferation assay, real time-polymerase chain reaction (RT-PCR) and immunoblotting were carried out to determine the effect of the transfections on cardiomyocyte proliferation, expressions of miR-24 and miR-34, as well as expressions of the target genes, TGF-β1 and E2F3.Results: The proliferation ability of the transfected cells was decreased significantly, relative to negative control. In contrast, percentage apoptosis was higher in the negative control group than in transfected cells. The expression profiles of anti-miR-24 and anti-miR-34 were significantly reduced, when compared to negative control. These results were further confirmed using immunoblot which revealed that the expression of the target genes, TGF-β1 and E2F3, were upregulated in the transfected cells.Conclusion: These results suggest that the synthetic oligonucleotides (anti-miR-24 and anti-miR-34) might be useful in developing therapeutic drug targets for cardiac diseases by suppressing the expressions of miR-24 and miR-34. Keywords: H9c2rat cardiomyocyte miRNA, Anti-miR-24, Anti-miR-34, Cardiac diseases