DNA-based Research Articles

Breaking genetic shackles: The advance of base editing in genetic disorder treatment.

The rapid evolution of gene editing technology has markedly improved the outlook for treating genetic diseases. Base editing, recognized as an exceptionally precise genetic modification tool, is emerging as a focus in the realm of genetic disease therapy. We provide a comprehensive overview of the fundamental principles and delivery methods of cytosine base editors (CBE), adenine base editors (ABE), and RNA base editors, with a particular focus on their applications and recent research advances in the treatment of genetic diseases. We have also explored the potential challenges faced by base editing technology in treatment, including aspects such as targeting specificity, safety, and efficacy, and have enumerated a series of possible solutions to propel the clinical translation of base editing technology. In conclusion, this article not only underscores the present state of base editing technology but also envisions its tremendous potential in the future, providing a novel perspective on the treatment of genetic diseases. It underscores the vast potential of base editing technology in the realm of genetic medicine, providing support for the progression of gene medicine and the development of innovative approaches to genetic disease therapy.

Enhancement of intrinsic guanine fluorescence by protonation in DNA of various structures

Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides.

RNA base editors: The emerging approach of RNA therapeutics.

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m6A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.

Significant downregulation of Alzheimer's amyloid-β levels enabled by engineered DNA nanomaterials

Although there are no effective therapies to block or reverse Alzheimer's disease (AD) progression at present, a promising therapeutic strategy is to reduce levels of amyloid-β (Aβ) proteins, which drive the formation of amyloid plaque, a primary hallmark in AD brains. Herein, we report that amphiphilic lipid-DNA molecules (LD) were designed by incorporating a long alkyl chain into the nucleotide base. It significantly down-regulated Alzheimer's Aβ levels in vivo and in vitro. In contrast to small-molecule chemical drugs and antibody therapies, the assembled DNA nanoparticles allowed them to effectively cross the blood-brain barrier (BBB) and accumulate in the brain, increasing the therapeutic effects. Notably, lipid-DNA downregulated the levels of Aβ peptides significantly in vitro. AD mice model experiments demonstrated that the LD-treated groups exhibited a rapid cognition behavioral improvement, which was associated with brain engagement of LD and reduced Aβ levels. Thus, the molecularly engineered DNA nanomaterials effectively regulated Aβ peptides. This work might provide a promising DNA engineering strategy for AD treatment.

IR and Raman markers of the interactions between MoS2 and pyrimidine bases

Changes in vibrational spectra are among the most important manifestations of the interaction between transition metal dichalcogenides and nucleic acid bases (NAB). Infrared and Raman spectra were calculated using the DFT/M06-2X method for the most stable stacked and covalently bonded complexes of pyrimidine bases with MoS2. Obtained spectra were analyzed to determine the spectral markers of the interactions between the pyrimidine bases and MoS2. We found that interaction with MoS2 leads to significant changes in both frequencies and intensities of NAB vibrations. The correlation between the changes and interaction energies of the NAB molecules with MoS2 was demonstrated. In general, changes in the vibrational frequencies in complexes with covalent bonds are significantly greater than ones in stacked ones. For the bonded complexes, the most significant changes are observed for those fragments of the NAB molecules that are directly involved in the formation of the covalent bonds. Calculations predict the decrease of the IR intensity and Raman activities of the NAB molecules in the spectra of stacked complexes and their increase in the spectra of covalently bonded complexes.

Differential effects of benzo[a]pyrene exposure on glutathione and purine metabolism in keratinocytes: Dose-dependent and UV co-exposure effects.

Polycyclic aromatic hydrocarbons with the key substance benzo[a]pyrene (B[a]P) are widespread pollutants in the environment and at working places. Nonetheless, the exact underlying mechanisms of toxicological effects caused by B[a]P especially in absence and presence of UV irradiation remain uncertain. This study examines variations in exposure conditions: low B[a]P (4 nM), low B[a]P + UV and high B[a]P (4 μM), selected based on pertinent cytotoxicity assessments. Following cell viability evaluations post-treatment with varied B[a]P concentrations and UV irradiation, the identified concentrations underwent detailed metabolomic analysis via gas chromatography-mass spectrometry. Subsequently, resulting changes in metabolic profiles across these distinct exposure groups are comprehensively compared. Chemometric analyses showed modest regulation of metabolites after low B[a]P exposure compared to control conditions. High B[a]P and low B[a]P + UV exposure significantly increased regulation of metabolic pathways, indicating that additional UV irradiation plus low B[a]P is as demanding for the cells as higher B[a]P treatment alone. Further analysis revealed exposure-dependent regulation of glutathione-important for oxidative defence-and purine metabolism-important for DNA base synthesis. Only after low B[a]P, oxidative defence appeared to be able to compensate for B[a]P-induced perturbations of the oxidative homeostasis. In contrast, purine metabolism already responded towards adversity at low B[a]P. The metabolomic results give an insight into the mechanisms leading to the toxic response and confirm the strong effects of co-exposure on oxidative defence and DNA repair in the model studied.

Regulatory activity is the default DNA state in eukaryotes.

Genomes encode for genes and non-coding DNA, both capable of transcriptional activity. However, unlike canonical genes, many transcripts from non-coding DNA have limited evidence of conservation or function. Here, to determine how much biological noise is expected from non-genic sequences, we quantify the regulatory activity of evolutionarily naive DNA using RNA-seq in yeast and computational predictions in humans. In yeast, more than 99% of naive DNA bases were transcribed. Unlike the evolved transcriptome, naive transcripts frequently overlapped with opposite sense transcripts, suggesting selection favored coherent gene structures in the yeast genome. In humans, regulation-associated chromatin activity is predicted to be common in naive dinucleotide-content-matched randomized DNA. Here, naive and evolved DNA have similar co-occurrence and cell-type specificity of chromatin marks, challenging these as indicators of selection. However, in both yeast and humans, extreme high activities were rare in naive DNA, suggesting they result from selection. Overall, basal regulatory activity seems to be the default, which selection can hone to evolve a function or, if detrimental, repress.

Programmable RNA base editing via targeted modifications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editors are powerful tools in biology and hold great promise for the treatment of human diseases. Advanced DNA base editing tools, such as cytosine base editor and adenine base editor, have been developed to correct permanent mistakes in genetic material. However, undesired off-target edits would also be permanent, which poses a considerable risk for therapeutics. Alternatively, base editing at the RNA level is capable of correcting disease-causing mutations but does not lead to lasting genotoxic effects. RNA base editors offer temporary and reversible therapies and have been catching on in recent years. Here, we summarize some emerging RNA editors based on A-to-inosine, C-to-U and U-to-pseudouridine changes. We review the programmable RNA-targeting systems as well as modification enzyme-based effector proteins and highlight recent technological breakthroughs. Finally, we compare editing tools, discuss limitations and opportunities, and provide insights for the future directions of RNA base editing.

DNA and RNA base editors can correct the majority of pathogenic single nucleotide variants

The majority of human genetic diseases are caused by single nucleotide variants (SNVs) in the genome sequence. Excitingly, new genomic techniques known as base editing have opened efficient pathways to correct erroneous nucleotides. Due to reliance on deaminases, which have the capability to convert A to I(G) and C to U, the direct applicability of base editing might seem constrained in terms of the range of mutations that can be reverted. In this evaluation, we assess the potential of DNA and RNA base editing methods for treating human genetic diseases. Our findings indicate that 62% of pathogenic SNVs found within genes can be amended by base editing; 30% are G>A and T>C SNVs that can be corrected by DNA base editing, and most of them by RNA base editing as well, and 29% are C>T and A>G SNVs that can be corrected by DNA base editing directed to the complementary strand. For each, we also present several factors that affect applicability such as bystander and off-target occurrences. For cases where editing the mismatched nucleotide is not feasible, we introduce an approach that calculates the optimal substitution of the deleterious amino acid with a new amino acid, further expanding the scope of applicability. As personalized therapy is rapidly advancing, our demonstration that most SNVs can be treated by base editing is of high importance. The data provided will serve as a comprehensive resource for those seeking to design therapeutic base editors and study their potential in curing genetic diseases.

No evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 nanoparticles in three mammalian cell lines despite the initial reduction of cellular mitochondrial activity

Abstract We studied the effects of Aeroxide P25 titanium dioxide nanoparticles (TiO2 NPs) with a diameter of 21 nm on induction of DNA damage and long-term survival of three human cell lines: hepatocellular liver carcinoma HepG2, colorectal adenocarcinoma HT29 and lung carcinoma A549. The endpoints examined were DNA breakage estimated by the comet assay and oxidative base damage recognized by formamide-pyrimidine glycosylase (FPG) estimated with the FPG+ comet assay, frequencies of histone γH2AX foci and micronuclei, apoptosis, cell metabolic activity measured by mitochondrial activity (MTT) assay and long-term survival measured by colony-forming ability. Each cell line had a different pattern of DNA breakage and base damage vs. nanoparticle (NP) concentration and treatment time. There was no increase in the frequencies of histone γH2AX foci and micronuclei as compared to those in the untreated cells. In parallel with these results, no induction of apoptosis has been found in none of the cell lines tested. The reported experiments provided no evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 NPs, despite a slight decrease in mitochondrial activity and cell survival during the first 72 h.

Melting and Bubble Formation in a Double-Stranded DNA: Microscopic Aspects of Early Base-Pair Opening Events and the Role of Water.

Despite its rigid structure, DNA is a remarkably flexible molecule. Flexibility is essential for biological functions (such as transcription and gene repair), which require large-amplitude structural changes such as bubble formation. The bubbles thus formed are required to have a certain stability of their own and survive long on the time scale of molecular motions. A molecular understanding of fluctuations leading to quasi-stable structures is not available. Through extensive atomistic molecular dynamics simulations, we identify a sequence of microscopic events that culminate in local bubble formation, which is initiated by base-pair (BP) opening, resulting from the cleavage of native BP hydrogen bonds (HBs). This is followed by the formation of mismatched BPs with non-native contacts. These metastable structures can either revert to their original forms or undergo a flipping transition to form a local bubble that can span across 3-4 BPs. A substantial distortion of the DNA backbone and a disruption of BP stacking are observed because of the structural changes induced by these local perturbations. We also explored how water helps in the entire process. A small number of water molecules undergo rearrangement to stabilize the intermediate states by forming HBs with DNA bases. Water thus acts as a lubricant that counteracts the enthalpic penalty suffered from the loss of native BP contacts. Although the process of bubble formation is reversible, the sequence of steps involved poses an entropic barrier, preventing it from easily retracing the path to the native state.

Exploring protein derivative profiles in cheese whey through native Candida tropicalis fermentation

ABSTRACT Native yeasts produce most peptides and amino acids from cheese whey fermentation. The research aimed to determine the protein, peptides, and amino acid profiles regarding whey fermentation by spontaneous fermentation and re-inoculation by native yeasts as a basis for determining potential yeasts in amino acid production. The selected native yeasts were identified through genome-based analysis on the DNA base sequence of the Internal Transcribed Spacer (ITS) region. The molecular identification shows that isolate CT1 is 95.62% comparable to Candida tropicalis strain VIT-NN02, CT2 is 98.85% same to stain NB5Y, and CT3 is 99.42% similar to GT 6. Whey fermentation was conducted by inoculating 3% each native C. tropicalis and without inoculation for spontaneous fermentation, then incubated for 48 hours at ambient temperature (26 ± 2°C). Unlike the other samples, the spontaneously fermented mozzarella whey was incubated without inoculation after cheese separation. After 48 hours, fermented whey was kept at 4°C for examination. The Kjeldahl method evaluated protein concentration, Folin-Ciocalteau determined peptide levels, and HPLC determined amino acid composition. C.tropicalis was identified as cheese whey native yeasts, and all treatments decreased protein and peptides while increased the total amino acid includes every single amino acid value except histidine during isolate CT1 whey fermentation. Candida tropicalis CT2 shown as potential isolate for the amino acid production from whey, with the protein contents of 0.846 ± 0.151%, protein decreases of 0.384 ± 0.186, peptide levels of 264.75 ± 40.40ppm, peptide decreases of 99.87 ± 37.96 and the highest total amino acid of 6149.56ppm.

Structure of native four-repeat satellite III sequence with non-canonical base interactions.

Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5'-ATGGAATGGA ATGGA ATGGA-3' using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05-0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G-G intercalation sandwiched by sheared A-G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A-T/T-A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

Classifying promoters by interpreting the hidden information of DNA sequences for disease prediction in clinical laboratories using Gaussian decision boundary estimation

A promoter is a brief stretch of DNA (100–1,000 bp) where RNA polymerase starts to transcribe a gene. A DNA (Deoxyribonucleic Acid) base pair is a fundamental unit of DNA structure and represents the pairing of two complementary nucleotide bases within the DNA double helix. The four DNA nucleotide bases are adenine (A), thymine (T), cytosine (C), and guanine (G). DNA base pairs are the building blocks of the DNA molecule, and their complementary pairing is central to the storage and transmission of genetic information in all living organisms. Normally, a promoter is found at the 5′ end of the transcription initiation site or immediately upstream. Numerous human disorders, particularly diabetes, cancer, and Huntington’s disease, have been shown to have DNA promoter as their root cause. The scientific community has long been interested in learning crucial information about protein-coding genes. Finding the promoters is therefore the first step in finding genes in DNA sequences. The scientific world has always been attracted by the effort to glean crucial knowledge about protein-coding genes. Consequently, identifying promoters has emerged as an intriguing challenge that has caught the interest of numerous researchers in the field of bioinformatics. We proposed Gaussian Decision Boundary Estimation in machine learning models to detect transcription start sites (promoters) in the DNA sequences of a common bacteria, Escherichia coli. The best features are identified through a score-based function to select relevant nucleotides that are directly responsible for promoter recognition, in order maximise the models’ performance. The Gaussian Decision Boundary Estimation based support-vector-machine model is trained with these features and finds the best hyperplane that separates the data into different classes. Throughout this study, promoter regions could be identified with high accuracy 99.9% which is better compared to other state of art algorithms. The comparison of machine learning classification models is another major emphasis of this paper in order to identify the model that most accurately predicts DNA sequence promoters. It provides analysis for further biological research as well as precision medicine.

RUBICON: a framework for designing efficient deep learning-based genomic basecallers

Nanopore sequencing generates noisy electrical signals that need to be converted into a standard string of DNA nucleotide bases using a computational step called basecalling. The performance of basecalling has critical implications for all later steps in genome analysis. Therefore, there is a need to reduce the computation and memory cost of basecalling while maintaining accuracy. We present RUBICON, a framework to develop efficient hardware-optimized basecallers. We demonstrate the effectiveness of RUBICON by developing RUBICALL, the first hardware-optimized mixed-precision basecaller that performs efficient basecalling, outperforming the state-of-the-art basecallers. We believe RUBICON offers a promising path to develop future hardware-optimized basecallers.

Photochemical processes to cellular DNA damage by UV radiation of different wavelengths: biological consequences

UV radiation of sunlight induces in cellular DNA of different organisms photochemical reactions, which may lead to the development of series biological responses to arising lesions, including apoptosis, mutagenesis, and carcinogenesis. The chemical nature and the amount of DNA lesions depend on the wavelength of UV radiation. Photons of UV radiation in the region B (UVB, 290–320 nm) cause the production of two main defects, namely, cyclobutane pyrimidine dimers and, with a less yield, pyrimidine (6-4) pyrimidone photoproducts; their formation is the result of the direct UVB photon absorption by DNA bases. Photons of UV radiation in the region A (UVA, 320–400 nm) induce only cyclobutane dimers that can be formed by triplet-triplet energy transfer from cellular chromophores, absorbing photons of this UV region, to DNA thymine bases. UVA is much more effective than UVB in the sensitized oxidatively lesion formation in DNA such as single strand breaks and oxidized bases; among those, 8-oxo-dihydroguanine is the most frequent since it can be produced from several oxidation processes. In recent years, multiple papers, reporting novel, more detailed information about molecular mechanisms of photochemical reactions underlying the formation of different lesions in DNA were published. The present review mainly aims at summarizing and analyzing data contained in these publications, particularly regarding oxidative reactions that are initiated by reactive oxygen species and radicals generated by potential endogenous photosensitizers such as pterins, riboflavin, protoporphyrin IX, NADH, and melanin. The role of specific DNA photoproducts in genotoxic processes induced in living systems by UV radiation of different wavelengths, including human skin carcinogenesis, is discussed.

Two new species of Erythroneurini (Hemiptera, Cicadellidae, Typhlocybinae) from southern China based on morphology and complete mitogenomes.

Erythroneurine leafhoppers (Hemiptera, Cicadellidae, Typhlocybinae, Erythroneurini) are utilized to resolve the relationship between the four erythroneurine leafhopper (Hemiptera, Cicadellidae, Typhlocybinae, Erythroneurini): Arboridia (Arboridia) rongchangensis sp. nov., Thaia (Thaia) jiulongensis sp. nov., Mitjaevia bifurcata Luo, Song & Song, 2021 and Mitjaevia diana Luo, Song & Song, 2021, the two new species are described and illustrated. The mitochondrial gene sequences of these four species were determined to update the mitochondrial genome database of Erythroneurini. The mitochondrial genomes of four species shared high parallelism in nucleotide composition, base composition and gene order, comprising 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and an AT control region, which was consistent with majority of species in Cicadellidae; all genes revealed common trait of a positive AT skew and negative GC skew. The mitogenomes of four species were ultra-conservative in structure, and which isanalogous to that of others in size and A + T content. Phylogenetic trees based on the mitogenome data of these species and another 24 species were built employing the maximum likelihood and Bayesian inference methods. The results indicated that the four species belong to the tribe Erythroneurini, M. diana is the sister-group relationship of M. protuberanta + M. bifurcata. The two species Arboridia (Arboridia) rongchangensis sp. nov. and Thaia (Thaia) jiulongensis sp. nov. also have a relatively close genetic relationship with the genus Mitjaevia.

Assessment of genetic diversity of freshwater genus Labeo through DNA barcoding and phylogenetic analysis inhabited in the River Indus

ObjectivesGenus Labeo of family Cyprinidae comprising of important group of fish species are known for ornamental purpose, food, extracting oil, medicinal value and for potential viable aquaculture. The present study was conducted to assess the morphometric and molecular identification of freshwater fishes of genus Labeo including Catla catla, Labeo rohita, Labeo calbasu, Labeo gnius, Labeo bata from the three fish resources of River Indus, Punjab, Pakistan. MethodsOut of 75, thirty fish specimen were amplified and sequenced with 650 bp of mitochondrial COI gene for the evaluation of fish biodiversity, management and conservation by using different bioinformatics techniques. ResultsMaximum and minimum genetic divergence between fishes of C. catla, L. calbasu, L. boga, L. gonius and L. rohita, were 0.052 to 0.105, respectively. Highest divergence was found between Labeo bata and Labeo rohita. Interspecific mean distance was found higher than intraspecific distance. In nucleotide base composition, AT contents was higher than GC contents indicated the clear pattern of anti-G bias. Transition rate was k1 = 8.84 for purines and transversional rate was k2 = 15.47 for pyrimidines with transition/transversion bias R = 5.62. Tajima D test and nucleotide diversity was measured D = 1.382883 and π = 0.049981 respectively. Positive value of D indicated a strong selective pressure and lack of population expansion. Thirty species were identified with e-value 0.0 and similar identity 99.85 %–100 % with maximum 600 bp length. Phylogenetic tree revealed two major clades with 95 % high bootstrap values identity showed closet relationship among these species. ConclusionsCurrent study supported the efficacy of the COI gene for the species identification in River Indus and develop the concept for establishing Pakistani fish gene bank which is still needed.

Using Graphdiyne Nanoribbons for Molecular Electronics Spectroscopy and Nucleobase Identification: A Theoretical Investigation.

In pursuit of fast, cost-effective, and reliable DNA sequencing techniques, a variety of two-dimensional (2D) material-based nanodevices such as solid-state nanopores and nanochannels have been explored and established. Given the promising potential of graphene for the design and fabrication of nanobiosensors, other 2D carbon allotropes such as graphyne and graphdiyne have also attracted a great deal of attention as candidate materials for the development of sequencing technology. Herein, employing the 2D electronic molecular spectroscopy (2DMES) method, we investigate the capability of graphdiyne nanoribbons (GDNRs) as the building blocks of a feasible, precise, and ultrafast sequencing device. Using first-principles calculations, we study the adsorption of four canonical nucleobases (NBs), i.e., adenine (A), cytosine (C), guanine (G), and thymine (T) on an armchair GDNR (AGDNR). Our calculations reveal that compared to graphene, graphdiyne demonstrates more distinct binding energies for different NBs, indicating its more promising ability to unambiguously recognize DNA bases. Utilizing the 2DMES technique, we calculate the differential conductance (Δg) of the studied NB-AGDNR systems and show that the resulting Δg maps, unique for each NB-AGDNR complex, can be used to recognize each individual NB without ambiguity. We also investigate the conductance sensitivity of the proposed nanobiosensor and show that it exhibits high sensitivity and selectivity toward various NBs. Thus, our proposed graphdiyne-based nanodevice would hold promise for next-generation DNA sequencing technology.

Effects of the spiroiminodihydantoin lesion on DNA base pair opening

Effects of the spiroiminodihydantoin lesion on DNA base pair opening