Nucleotide Context Research Articles

The influence of nucleotide context on non-specific amplification of DNA with Bst exo- DNA polymerase

In recent years, nucleic acid amplification methods that proceed in isothermal mode and require the use of DNA polymerases with strand-displacement activity have become increasingly widespread. Among these, the most popular is the Bst exo- polymerase, but it tends to carry out nonspecific DNA synthesis through multimerization. This study shows the influence of the nucleotide sequence on the binding of Bst exo- with DNA and on the efficiency of multimerization initiation. On single-stranded trinucleotides (sst) and dinucleotide duplexes (dst), molecular docking revealed the preference for binding of the “closed” form of Bst exo- to purine-rich sequences, especially those containing dG at the 3′ end of the synthesized strand. The data obtainedin silicowere confirmed in experiments using oligonucleotide templates that differ in the structure of the 3′- and 5′-terminal motifs. It has been shown that templates with an oligopurine 3′-terminal fragment and an oligopyrimidine 5′-terminal part contribute to an earlier start of multimerization. The obtained data can be used at the stage of selecting optimal nucleotide sequences for isothermal amplification, ensuring more reliable results. Thus, to avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

D-sORF: Accurate Ab Initio Classification of Experimentally Detected Small Open Reading Frames (sORFs) Associated with Translational Machinery.

Small open reading frames (sORFs; <300 nucleotides or <100 amino acids) are widespread across all genomes, and an increasing variety of them appear to be translating from non-genic regions. Over the past few decades, peptides produced from sORFs have been identified as functional in various organisms, from bacteria to humans. Despite recent advances in next-generation sequencing and proteomics, accurate annotation and classification of sORFs remain a rate-limiting step toward reliable and high-throughput detection of small proteins from non-genic regions. Additionally, the cost of computational methods utilizing machine learning is lower than that of biological experiments, and they can be employed to detect sORFs, laying the groundwork for biological experiments. We present D-sORF, a machine-learning framework that integrates the statistical nucleotide context and motif information around the start codon to predict coding sORFs. D-sORF scores directly for coding identity and requires only the underlying genomic sequence, without incorporating parameters such as the conservation, which, in the case of sORFs, may increase the dispersion of scores within the significantly less conserved non-genic regions. D-sORF achieves 94.74% precision and 92.37% accuracy for small ORFs (using the 99 nt medium length window). When D-sORF is applied to sORFs associated with ribosomes, the identification of transcripts producing peptides (annotated by the Ensembl IDs) is similar to or superior to experimental methodologies based on ribosome-sequencing (Ribo-Seq) profiling. In parallel, the recognition of putative negative data, such as the intron-containing transcripts that associate with ribosomes, remains remarkably low, indicating that D-sORF could be efficiently applied to filter out false-positive sORFs from Ribo-Seq data because of the non-productive ribosomal binding or noise inherent in these protocols.

Exploring the impact of sequence context on errors in SNP genotype calling with whole genome sequencing data using AI-based autoencoder approach.

A critical step in the analysis of whole genome sequencing data is variant calling. Despite its importance, variant calling is prone to errors. Our study investigated the association between incorrect single nucleotide polymorphism (SNP) calls and variant quality metrics and nucleotide context. In our study, incorrect SNPs were defined in 20 Holstein-Friesian cows by comparing their SNPs genotypes identified by whole genome sequencing with the IlluminaNovaSeq6000 and the EuroGMD50K genotyping microarray. The dataset was divided into the correct SNP set (666 333 SNPs) and the incorrect SNP set (4 557 SNPs). The training dataset consisted of only the correct SNPs, while the test dataset contained a balanced mix of all the incorrectly and correctly called SNPs. An autoencoder was constructed to identify systematically incorrect SNPs that were marked as outliers by a one-class support vector machine and isolation forest algorithms. The results showed that 59.53% (±0.39%) of the incorrect SNPs had systematic patterns, with the remainder being random errors. The frequent occurrence of the CGC 3-mer was due to mislabelling a call for C. Incorrect T instead of A call was associated with the presence of T in the neighbouring downstream position. These errors may arise due to the fluorescence patterns of nucleotide labelling.

Environment modulates protein heterogeneity through transcriptional and translational stop codon readthrough

Stop codon readthrough events give rise to longer proteins, which may alter the protein’s function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Properties and Mechanisms of Deletions, Insertions, and Substitutions in the Evolutionary History of SARS-CoV-2.

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.

Epigenetic editing for autosomal dominant neurological disorders.

Epigenetics refers to the molecules and mechanisms that modify gene expression states without changing the nucleotide context. These modifications are what encode the cell state during differentiation or epigenetic memory in mitosis. Epigenetic modifications can alter gene expression by changing the chromatin architecture by altering the affinity for DNA to wrap around histone octamers, forming nucleosomes. The higher affinity the DNA has for the histones, the tighter it will wrap and therefore induce a heterochromatin state, silencing gene expression. Several groups have shown the ability to harness the cell's natural epigenetic modification pathways to engineer proteins that can induce changes in epigenetics and consequently regulate gene expression. Therefore, epigenetic modification can be used to target and treat disorders through the modification of endogenous gene expression. The use of epigenetic modifications may prove an effective path towards regulating gene expression to potentially correct or cure genetic disorders.

Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo- DNA Polymerase.

Isothermal nucleic acids amplification that requires DNA polymerases with strand-displacement activity gained more attention in the last two decades. Among the DNA polymerases with strand-displacement activity, Bst exo- is the most widely used. However, it tends to carry out nonspecific DNA synthesis through multimerization. In this study, the effect of nucleotide sequence on the Bst exo- binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the "closed" form of Bst exo- to the purine-rich DNA sequences, especially those containing dG at the 3'-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtained in silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3'- and 5'-terminal motifs. It has been shown that templates with the oligopurine 3'-terminal fragment and oligopyrimidine 5'-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplification. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

A molnupiravir-associated mutational signature in global SARS-CoV-2 genomes

Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load1,2. However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir.

Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

The small nuclear RNAs 4.5SH and 4.5SI were characterized only in mouse-like rodents; their genes originate from 7SL RNA and tRNA, respectively. Similar to many genes transcribed by RNA polymerase III (pol III), the genes of 4.5SH and 4.5SI RNAs include boxes A and B, forming an intergenic pol III-directed promoter. In addition, their 5'-flanking sequences have TATA-like boxes at position -31/-24, also required for efficient transcription. The patterns of the three boxes notably differ in the 4.5SH and 4.5SI RNA genes. The A, B, and TATA-like boxes were replaced in the 4.5SH RNA gene with the corresponding boxes in the 4.5SI RNA gene to evaluate their effect on the transcription of transfected constructs in HeLa cells. Simultaneous replacement of all three boxes decreased the transcription level by 40%, which indicates decreased promoter activity in a foreign gene. We developed a new approach to compare the promoter strength based on the competition of two co-transfected gene constructs when the proportion between the constructs modulates their relative activity. This method demonstrated that the promoter activity of 4.5SI is 12 times that of 4.5SH. Unexpectedly, the replacement of all three boxes of the weak 4.5SH promoter with those of the strong 4.5SI gene significantly reduced, rather than enhanced, the promoter activity. Thus, the strength of a pol III-directed promoter can depend on the nucleotide environment of the gene.

Dynamics of 8-Oxoguanine in DNA: Decisive Effects of Base Pairing and Nucleotide Context.

8-Oxo-7,8-dihydroguanine (oxoG), an abundant DNA lesion, can mispair with adenine and induce mutations. To prevent this, cells possess DNA repair glycosylases that excise either oxoG from oxoG:C pairs (bacterial Fpg, human OGG1) or A from oxoG:A mispairs (bacterial MutY, human MUTYH). Early lesion recognition steps remain murky and may include enforced base pair opening or capture of a spontaneously opened pair. We adapted the CLEANEX-PM NMR protocol to detect DNA imino proton exchange and analyzed the dynamics of oxoG:C, oxoG:A, and their undamaged counterparts in nucleotide contexts with different stacking energy. Even in a poorly stacking context, the oxoG:C pair did not open easier than G:C, arguing against extrahelical base capture by Fpg/OGG1. On the contrary, oxoG opposite A significantly populated the extrahelical state, which may assist recognition by MutY/MUTYH.

LRT-CLUSTER: A New Clustering Algorithm Based on Likelihood Ratio Test to Identify Driving Genes.

Somatic mutations often occur at high relapse sites in protein sequences, which indicates that the location clustering of somatic missense mutations can be used to identify driving genes. However, the traditional clustering algorithm has such problems as the background signal over-fitting, the clustering algorithm is not suitable for mutation data, and the performance of identifying low-frequency mutation genes needs to be improved. In this paper, we propose a linear clustering algorithm based on likelihood ratio test knowledge to identify driver genes. In this experiment, firstly, the polynucleotide mutation rate is calculated based on the prior knowledge of likelihood ratio test. Then, the simulation data set is obtained through the background mutation rate model. Finally, the unsupervised peak clustering algorithm is used to, respectively, evaluate the somatic mutation data and the simulation data to identify the driver genes. The experimental results show that our method achieves a better balance of precision and sensitivity. It can also identify the driver genes missed by other methods, making it an effective supplement to other methods. We also discover some potential linkages between genes and between genes and mutation sites, which is of great value to target drug therapy research. Method framework: Our proposed model framework is as follows. a. Counting mutation sites and the number of mutations in tumor gene elements. b. The nucleotide context mutation frequency is counted based on the likelihood ratio test knowledge, and the background mutation rate model is obtained. c. Based on Monte Carlo simulation method, data sets with the same number of mutations as gene elements are randomly sampled to obtain simulated mutation data, and the sampling frequency of each mutation site is related to the mutation rate of polynucleotide. d. The original mutation data and the simulated mutation data after random reconstruction are clustered by peak density, respectively, and the corresponding clustering scores are obtained. e. We can obtain the clustering information statistics in each gene segment and score of each gene segment from the original single nucleotide mutation data through step d. f. According to the observed score and the simulated clustering score, the p-value of the corresponding gene fragment is calculated. g. We can obtain the clustering information statistics in each gene segment and score of each gene segment from the simulated single nucleotide mutation data through step d.

The context signals of mitochondrial miRNAs (mitomiRs) of mammals.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level in the cytoplasm and play an important role in a wide range of biological processes. Recent studies have found that the miRNA sequences are presented not only in the cytoplasm, but also in the mitochondria. These miRNAs (the so-called mitomiRs) may be the sequences of nuclear or mitochondrial origin; some of them are involved in regulation of the mitochondrial gene functions, while the role of others is still unknown. The identification of nucleotide signals, which are unique to mitomiRs, may help to determine this role. We formed a dataset that combined the experimentally discovered mitomiRs in human, rat and mouse. To isolate signals that may be responsible for the mitomiRs' functions or for their translocation from or into mitochondria a context analysis was carried out for the sequences. For three species in the group mitomiRs/non-mitomiRs and the group of all miRNAs from the miRBase database statistically overrepresented 8-letter motifs were identified (p-value < 0.01 with Bonferroni correction for multiple comparisons), for these motifs the patterns of the localization in functionally important regions for different types of miRNAs were found. Also, for the group mitomiRs/non-mitomiRs we found the statistically significant features of the miRNA nucleotide context near the Dicer and Drosha cleavage sites (Pearson's χ2 test of independence for the first three positions of the miRNA, p-value < 0.05). The observed nucleotide frequencies may indicate a more homogeneous pri-miRNA cleavage by the Drosha complex during the formation of the 5' end of mitomiRs. The obtained results can help to determine the role of the nucleotide signals in the origin, processing, and functions of the mitomiRs.

Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts.

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.

Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts

Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts

Improving language model of human genome for DNA-protein binding prediction based on task-specific pre-training.

The DNA-protein binding plays a pivotal role in regulating gene expression and evolution, and computational identification of DNA-protein has drawn more and more attention in bioinformatics. Recently, variants of BERT are also used to capture the semantic information of DNA sequences for predicting DNA-protein bindings. In this study, we leverage a task-specific pre-training strategy on BERT using large-scale multi-source DNA-protein binding data and present TFBert. TFBert treats DNA sequences as natural sentences and k-mer nucleotides as words. It can effectively extract upstream and downstream nucleotide context information by pre-training the 690 unlabeled ChIP-seq datasets. Experiments show that the pre-trained model can achieve promising performance on every single dataset in the 690 ChIP-seq datasets after simple fine tuning, especially on small datasets. The average AUC is 94.7%, outperforming existing popular methods. In conclusion, this study provides a variant of BERT based on pre-training and achieved state-of-the-art results in predicting DNA-protein bindings. We believe that TFBert can provide insights into other biological sequence classification problems.

Evolution, epidemiology, geographical distribution, and mutational landscape of newly emerging monkeypox virus.

Recent monkeypox (MPX) outbreaks are major ones in non-endemic countries. The present study analyzed molecular phylogenetics, divergence, epidemiology, the geographical distribution, entropy diversity of genome, mutational landscape, and evolution of the monkeypox virus (MPXV) genome and the current MPXV is entitled “hMPXV1.” We used different in-silico and statistical methods to study our objectives. The developed phylogram from molecular phylogenetics describes the origin and evolution of hMPXV1 of A, A.1, A.1.1, A.2, and B.1 lineages. The microevolution of B.1 lineage shows its evolution from May to August 2022. B.1 lineage is further adapting and showing more mutation and sub-lineages. The scatter plot of all lineages shows the clustering pattern of lineages and the divergence. We also developed two statistical models of confirmed cases and a diagram of the age-related pattern of infected cases to illustrate the epidemiology of the MPX outbreaks. The entropy diversity and mutational landscape of the hMPXV1 genome were analyzed in nucleotide and codon contexts. Our study has shown the in-depth evolution pattern of different lineages of the hMPXV1. We found B.1 lineage is associated with the current outbreaks. The mutational landscape informs about the slow mutation of the virus. Finally, the study might assists the new therapeutic development considering all the above points and would help the researcher to set up their future research directions.

Abstract 6262: Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide

Abstract The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient (MMRp) and unresponsive to immunotherapy, while MMR deficient (MMRd) tumors often respond to immune checkpoint blockade (ICB). We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB) and, sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR proficient and KRAS mutant mCRC patients receive priming therapy with TMZ. Analysis of solid tissue biopsies and circulating tumor DNA (ctDNA) obtained after TMZ treatment revealed the emergence of TMZ mutational signature, alterations in MMR genes and increased TMB in 14 out of 16 patients. Genetic mutations induced by TMZ were dose-dependent and multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes such as the MSH6 T1219I variant which was detected in ctDNA and tissue of 13/14 (93%) of the cases. A subset of the patients whose tumors after TMZ priming displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment. Overall, we provide proof-of-concept that treatment of MGMT deficient/MMR proficient KRAS mutant mCRCs with TMZ can be tracked by mutational signature analysis and lead to inactivation of the MMR pathway, emergence of the TMZ mutational signature, TMB increase, and, in some cases, to disease stabilization during ICB. Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Alessio Amatu, Marco Macagno, Ludovic Barault, Andrea Cassingena, Alice Bartolini, Paolo Luraghi, Gianluca Mauri, Paolo Battuello, Nicola Personemi, Valeria Pessei, Pietro Paolo Vitiello, Federica Tosi, Laura Idotta, Emanuele Valtorta, Emanuela Bonoldi, Giovanni Germano, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6262.

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.

Identification of lung cancer drivers by comparison of the observed and the expected numbers of missense and nonsense mutations in individual human genes.

Largely, cancer development is driven by acquisition and positive selection of somatic mutations that increase proliferation and survival of tumor cells. As a result, genes related to cancer development tend to have an excess of somatic mutations in them. An excess of missense and/or nonsense mutations in a gene is an indicator of its cancer relevance. To identify genes with an excess of potentially functional missense or nonsense mutations one needs to compare the observed and expected numbers of mutations in the gene. We estimated the expected numbers of missense and nonsense mutations in individual human genes using (i) the number of potential sites for missense and nonsense mutations in individual transcripts and (ii) histology-specific nucleotide context-dependent mutation rates. To estimate mutation rates defined as the number of mutations per site per tumor we used silent mutations reported in the Catalog Of Somatic Mutations In Cancer (COSMIC). The estimates were nucleotide context dependent. We have identified 26 genes with an excess of missense and/or nonsense mutations for lung adenocarcinoma, 18 genes for small cell lung cancer, and 26 genes for squamous cell carcinoma of the lung. These genes include known genes and novel lung cancer gene candidates.

Translation termination codons in protein synthesis and disease.

Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 3' of mRNA and drive the release of the polypeptide chain. However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.