Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

Comparison of Alternative Splicing Landscapes Revealed by Long-Read Sequencing in Hepatocyte-Derived HepG2 and Huh7 Cultured Cells and Human Liver Tissue.

The long-read RNA sequencing developed by Oxford Nanopore Technologies provides a direct quantification of transcript isoforms, thereby making it possible to present alternative splicing (AS) profiles as arrays of single splice variants with different abundances. Additionally, AS profiles can be presented as arrays of genes characterized by the degree of alternative splicing (the DAS-the number of detected splice variants per gene). Here, we successfully utilized the DAS to reveal biological pathways influenced by the alterations in AS in human liver tissue and the hepatocyte-derived malignant cell lines HepG2 and Huh7, thus employing the mathematical algorithm of gene set enrichment analysis. Furthermore, analysis of the AS profiles as abundances of single splice variants by using the graded tissue specificity index τ provided the selection of the groups of genes expressing particular splice variants specifically in liver tissue, HepG2 cells, and Huh7 cells. The majority of these splice variants were translated into proteins products and appeal to be in focus regarding further insights into the mechanisms underlying cell malignization. The used metrics are intrinsically suitable for transcriptome-wide AS profiling using long-read sequencing.

Principal Component Analysis of Alternative Splicing Profiles Revealed by Long-Read ONT Sequencing in Human Liver Tissue and Hepatocyte-Derived HepG2 and Huh7 Cell Lines.

The long-read RNA sequencing developed by Oxford Nanopore Technology provides a direct quantification of transcript isoforms. That makes the number of transcript isoforms per gene an intrinsically suitable metric for alternative splicing (AS) profiling in the application to this particular type of RNA sequencing. By using this simple metric and recruiting principal component analysis (PCA) as a tool to visualize the high-dimensional transcriptomic data, we were able to group biospecimens of normal human liver tissue and hepatocyte-derived malignant HepG2 and Huh7 cells into clear clusters in a 2D space. For the transcriptome-wide analysis, the clustering was observed regardless whether all genes were included in analysis or only those expressed in all biospecimens tested. However, in the application to a particular set of genes known as pharmacogenes, which are involved in drug metabolism, the clustering worsened dramatically in the latter case. Based on PCA data, the subsets of genes most contributing to biospecimens' grouping into clusters were selected and subjected to gene ontology analysis that allowed us to determine the top 20 biological processes among which translation and processes related to its regulation dominate. The suggested metrics can be a useful addition to the existing metrics for describing AS profiles, especially in application to transcriptome studies with long-read sequencing.

Identifying N6-Methyladenosine Sites in HepG2 Cell Lines Using Oxford Nanopore Technology.

RNA modifications, particularly N6-methyladenosine (m6A), are pivotal regulators of RNA functionality and cellular processes. We analyzed m6A modifications by employing Oxford Nanopore technology and the m6Anet algorithm, focusing on the HepG2 cell line. We identified 3968 potential m6A modification sites in 2851 transcripts, corresponding to 1396 genes. A gene functional analysis revealed the active involvement of m6A-modified genes in ubiquitination, transcription regulation, and protein folding processes, aligning with the known role of m6A modifications in histone ubiquitination in cancer. To ensure data robustness, we assessed reproducibility across technical replicates. This study underscores the importance of evaluating algorithmic reproducibility, especially in supervised learning. Furthermore, we examined correlations between transcriptomic, translatomic, and proteomic levels. A strong transcriptomic-translatomic correlation was observed. In conclusion, our study deepens our understanding of m6A modifications' multifaceted impacts on cellular processes and underscores the importance of addressing reproducibility concerns in analytical approaches.

Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

The 2'-deoxyuridine-5'-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (Ep) of 0.6-0.7 V and 0.8-0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between -0.9 V and -1 V, but they did not depend on concentration. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peaks at 0.8-0.9 V. In addition, the distinct reduction peaks at Ep between -0.8 V and -0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50-90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon output and labeling was achieved at the dUTP-Fl : dTTP and dUTP-Rh : dTTP molar ratios of 70% : 30% and 20% : 80% in the PCR mixture, respectively, allowing the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein and rhodamine modified dUTP appear as promising electroactive labels to develop direct electrochemical DNA assays for detecting PCR and RPA products.

Oxford Nanopore MinION Direct RNA-Seq for Systems Biology.

Long-read direct RNA sequencing developed by Oxford Nanopore Technologies (ONT) is quickly gaining popularity for transcriptome studies, while fast turnaround time and low cost make it an attractive instrument for clinical applications. There is a growing interest to utilize transcriptome data to unravel activated biological processes responsible for disease progression and response to therapies. This trend is of particular interest for precision medicine which aims at single-patient analysis. Here we evaluated whether gene abundances measured by MinION direct RNA sequencing are suited to produce robust estimates of pathway activation for single sample scoring methods. We performed multiple RNA-seq analyses for a single sample that originated from the HepG2 cell line, namely five ONT replicates, and three replicates using Illumina NovaSeq. Two pathway scoring methods were employed-ssGSEA and singscore. We estimated the ONT performance in terms of detected protein-coding genes and average pairwise correlation between pathway activation scores using an exhaustive computational scheme for all combinations of replicates. In brief, we found that at least two ONT replicates are required to obtain reproducible pathway scores for both algorithms. We hope that our findings may be of interest to researchers planning their ONT direct RNA-seq experiments.

Human Chr18 transcriptome dataset combined from the Illumina HiSeq, ONT MinION, and qPCR data.

The chromosome-centric dataset was created by applying several technologies of transcriptome profiling. The described dataset is available at NCBI repository (BioProject ID PRJNA635536). The dataset referred to the same type of tissue, cell lines, transcriptome sequencing technologies, and was accomplished in a period of 8 years (the first data were obtained in 2013 while the last ones - in 2020). The high-throughput sequencing technologies were employed along with the quantitative PCR (qPCR) approach, for data generation using the gene expression level assessment. qPCR was performed for a limited group of genes, encoded on human chromosome 18, for the Russian part of the Chromosome-Centric Human Proteome Project. The data of high-throughput sequencing are provided as Excel spreadsheets, where the data on FPKM and TMP values were evaluated for the whole transcriptome with both Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing.

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma.

Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized on magnetic beads. It was found that the aptamer-based enrichment provided a 30-fold increase in the sensitivity of SRM detection of SMAD4. These results indicate that the aptamer-based affinity enrichment of target proteins can be successfully employed to improve quantitative detection of low-abundance proteins by SRM in undepleted human blood plasma.

Chromosome 18 transcriptoproteome of liver tissue and HepG2 cells and targeted proteome mapping in depleted plasma: update 2013.

We report the results obtained in 2012-2013 by the Russian Consortium for the Chromosome-centric Human Proteome Project (C-HPP). The main scope of this work was the transcriptome profiling of genes on human chromosome 18 (Chr 18), as well as their encoded proteome, from three types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma. The transcriptome profiling for liver tissue was independently performed using two RNaseq platforms (SOLiD and Illumina) and also by droplet digital PCR (ddPCR) and quantitative RT-PCR. The proteome profiling of Chr 18 was accomplished by quantitatively measuring protein copy numbers in the three types of biomaterial (the lowest protein concentration measured was 10(-13) M) using selected reaction monitoring (SRM). In total, protein copy numbers were estimated for 228 master proteins, including quantitative data on 164 proteins in plasma, 171 in the HepG2 cell line, and 186 in liver tissue. Most proteins were present in plasma at 10(8) copies/µL, while the median abundance was 10(4) and 10(5) protein copies per cell in HepG2 cells and liver tissue, respectively. In summary, for liver tissue and HepG2 cells a "transcriptoproteome" was produced that reflects the relationship between transcript and protein copy numbers of the genes on Chr 18. The quantitative data acquired by RNaseq, PCR, and SRM were uploaded into the "Update_2013" data set of our knowledgebase (www.kb18.ru) and investigated for linear correlations.