Single Adenine Research Articles

Facile constructing of interlocking structure for improving anti-corrosion performance of adenine film on copper

A nanometer-thick adenine (Ade/NaClO) film with interlocking structure was successfully prepared by regulating the micro-etching process of sodium hypochlorite and the adsorption process of adenine. Compared to single adenine film, the compactness, adhesion and anti-corrosion performance of Ade/NaClO film were obviously improved. Electrochemical tests demonstrated that the corrosion protection efficiency was improved from 85.8 % to 98.2 % and exhibited self-strengthen anti-corrosion performance. In-situ Raman spectroscopy indicated that the oxidative etching effect of NaClO played an important role in the formation of the film. The strategy proposed in this study exhibits guiding significance for improving the performance of conversion films on metal.

Solvolysis of 2’-deoxyribonucleosides and oligo-2’-deoxyribonucleotides in aqueous pyrrolidine or ammonia solutions

To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the 2’-β-D-deoxyribosides of 5-bromouracil, 2-aminopurine, uracil, adenine, cytosine, 5-methylcytosine, hypoxanthine, N6-methyladenine, N4-ethylcytosine, and guanine were compared as to their rate of degradation in 0.5 M aqueous pyrrolidine at 110 °C, conditions used earlier in the analysis of oligonucleotides containing only the canonical bases. The reaction mixtures were analyzed by chromatography on Zorbax XDB-CN and UV absorption spectroscopy. The first-order rate constants for the nucleoside degradations decreased in the above order, spanning a wide range of reactivities. Some of these nucleosides were also tested in 0.5 M aqueous ammonia at 110 °C, giving similar first-order rate constants, except for 2’-deoxyguanosine, which is much more reactive with ammonia, due to the lower basicity of this reagent, leaving a larger proportion of the nucleoside in the non-ionized form, susceptible to nucleophilic attack at the base. Short oligothymidylates containing a single 2-aminopurine, adenine, guanine, or cytosine unit in central position were tested in pyrrolidinolysis, to determine the cleavage rates at these sites and the dependence of these cleavage rates on oligonucleotide length. A model decadeoxyribonucleotide containing all four canonical bases was also pyrrolidinolyzed, followed by ion-exchange chromatography, to deduce the nucleotide sequence from the resulting chromatographic profile.

Molecular doping of nucleic acids into light emitting crystals driven by multisite-intermolecular interaction

We reveal the fundamental understanding of molecular doping of DNAs into organic semiconducting tris (8-hydroxyquinoline) aluminum (Alq3) crystals by varying types and numbers of purines and pyrimidines constituting DNA. Electrostatic, hydrogen bonding, and π-π stacking interactions between Alq3 and DNAs are the major factors affecting the molecular doping. Longer DNAs induce a higher degree of doping due to electrostatic interactions between phosphate backbone and Alq3. Among four bases, single thymine bases induce the multisite interactions of π-π stacking and hydrogen bonding with single Alq3, occurring within a probability of 4.37%. In contrast, single adenine bases form multisite interactions, within lower probability (1.93%), with two-neighboring Alq3. These multisite interactions facilitate the molecular doping into Alq3 particles compared to cytosines or guanines only forming π-π stacking. Thus, photoluminescence and optical waveguide phenomena of crystals were successfully tailored. This discovery should deepen our fundamental understanding of incorporating DNAs into organic semiconducting crystals.

A novel shiga based immunotoxin against Fn-14 receptor on colorectal and lung cancer

Immunotoxins are regarded as a type of targeted therapy for killing cells by highly potent bacterial, fungal or plant toxins. Shiga like toxins (SLTs) are a group of bacterial AB5 protein toxins that inhibit host cell protein synthesis through the removal of a single adenine residue from the 28S rRNA and lead to apoptosis. Here, we described the design and usage of a Stx-based immunotoxin that can induce the selective cytotoxicity and apoptosis in Fn-14-positive cells related to the colon and lung cancer. In the present study, the Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system when driven from inclusion bodies by 8 M urea. The Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system and then purified. The purified fusion protein could specifically target Fn-14 receptor existed on colon and lung cancer cell lines and suppress these cells in a dose-dependent manner. In addition, the protein was able to nearly 50 % of apoptotic cell death and maintains about 54 % of its stability after 24 h of incubation in mouse serum at 37 °C. Compared to PE38-P4A8 construct in our previous study, these results showed that the Stx2a-PE15-P4A8 construct can be an efficient therapeutic candidate for cancer immunotherapy.

Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates.

Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427 motif I; L475Q, 7 aa upstream of the 482SDN484 motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622 motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes.

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.

Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

White noise functional integral for exponentially decaying memory: nucleotide distribution in bacterial genomes

We utilize a stochastic functional integral approach that forms a natural framework for analyzing ubiquitous complex sequences of fluctuations with underlying non-Markovian stochastic process beyond fractional Brownian motion. We demonstrate how Hida white noise calculus, guided by mean square deviation (MSD) analysis of empirical data, allows derivation of single nucleotide occurrence probability distributions for whole genomes of four significant species of bacteria: (a) freshwater cyanobacteria Synechococcus elongatus PCC7942, 2.7 Mbp, (b) marine cyanobacteria Prochlorococcus marinus subsp. marinus str. CCMP1375, 1.8 Mbp, (c) pathogenic bacteria Staphylococcus aureus subsp. aureus NCTC 8325, 2.8 Mbp, and (d) Staphylococcus aureus ILRI Eymole1/1, 2.9 Mbp. Here, the stochastic variable is chosen to represent separation distances between succeeding identical single nucleotides where distance is defined as the number of steps through intervening bases. The stochastic parameter set takes values of nucleotide occurrence count along the genome length. The probability density function (PDF) is derived in closed form for the associated stochastic process with exponentially damped memory kernel, and is shown to satisfy a modified diffusion equation with a parameter-dependent diffusion coefficient. The PDF yields an analytical result for MSDs that match empirical plots, showing a rising nonlinear curve that flattens to a plateau starting close to 1 kb, similar to restricted diffusion. The plots exhibit compliance with Chargaff’s second parity rule for nucleotides. The same PDF describes occurrences of single nucleotides adenine, guanine, cytosine, and thymine for all four bacterial genomes considered.

Gld2 activity is regulated by phosphorylation in the N-terminal domain

ABSTRACTThe de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling.

How Ricin Damages the Ribosome.

Ricin belongs to the group of ribosome-inactivating proteins (RIPs), i.e., toxins that have evolved to provide particular species with an advantage over other competitors in nature. Ricin possesses RNA N-glycosidase activity enabling the toxin to eliminate a single adenine base from the sarcin-ricin RNA loop (SRL), which is a highly conserved structure present on the large ribosomal subunit in all species from the three domains of life. The SRL belongs to the GTPase associated center (GAC), i.e., a ribosomal element involved in conferring unidirectional trajectory for the translational apparatus at the expense of GTP hydrolysis by translational GTPases (trGTPases). The SRL represents a critical element in the GAC, being the main triggering factor of GTP hydrolysis by trGTPases. Enzymatic removal of a single adenine base at the tip of SRL by ricin blocks GTP hydrolysis and, at the same time, impedes functioning of the translational machinery. Here, we discuss the consequences of SRL depurination by ricin for ribosomal performance, with emphasis on the mechanistic model overview of the SRL modus operandi.

Exciton Trapping Dynamics in DNA Multimers.

Using as a model the single adenine strand (dA)20, we study the ultrafast evolution of electronic excitations in DNA with a time resolution of 30 fs. Our transient absorption spectra in the UV and visible spectral domains show that internal conversion among photogenerated exciton states occurs within 100 fs. Subsequently, the ππ* states acquire progressively charge-transfer character before being completely trapped, within 3 ps, by fully developed charge-transfer states corresponding to transfer of an electron from one adenine moiety to another (A+A-).

Kinetic Analysis of Single Molecule Electrodiffusion in a Biological Nanopore with Two Binding Sites

When single short adenine oligonucleotides (d.p. 3-5) block the pore formed by the bacterial toxin aerolysin, the resultant resistive pulses are complex with two levels (short and deep vs. long and shallow) in a variety of configurations, the probability of which depends on voltage. We account for this by a four-state kinetic mechanism with two undistinguishable open states i,o and two blocked states m1 (short-deep) and m2 (long-shallow) linked as o<->m1<->m2->i. M1 is directly accessible from o which designates the presence of the nucleotide at the pore′s cis-side mouth, while m2 is inaccessible from state i, as nucleotides are unable to enter from the trans-side irrespective of voltage. In this framework, several experimentally accessible statistical quantities such as the frequency or probability of returns to m1 from m2, the fraction of restive pulses ending in m1 and the mean dwell times in m2 as well as the mean total duration of resistive pulses acquire mechanistic significance and allow direct kinetic predictions using a Q-matrix approach. For A3, the data are consistent with a charged particle moving through a one-dimensional energy landscape with two minimain an electrically biased random walk. For longer nucleotides (e.g. A5) the success rate for translocation is higher than predicted by the rate constants determined from the other observables. It appears likely that this is due to an ability of the longer oligomers to simultaneously link with the two binding sites, producing an excess of returns from m2 to m1, which, however, does not entail a propensity to result in translocation failures. Such double tethering of DNA might promote translocation of longer chains by producing an extended conformation and may also contribute to the strong rectification of transport observed for aerolysin.

Single-molecule spectroscopy of radical pairs, a theoretical treatment and experimental considerations

ABSTRACTIn this work, we propose to extend the scope of single-molecule spectroscopy to spin chemistry investigations of single radical pairs. A consistent theory of single-molecule spectroscopy on single radical pairs is proposed, which allows one to show that bunching phenomena are affected by singlet–triplet interconversion in radical pairs, which is, in turn, affected by local and external magnetic fields. A detailed study on the feasibility of such experiments with single flavin adenine dinucleotide, FAD, molecules is presented. We conclude that the observation of magnetic field effects on single FAD molecules is feasible but experimentally challenging for measurements on integrated fluorescence intensity and fluorescence event statistics.

Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions).

Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications.

Ribosome Inactivating Proteins from an evolutionary perspective

Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosidases that inhibit protein synthesis through the elimination of a single adenine residue from 28S rRNA. Many of these toxins have been characterized in depth from a biochemical and molecular point of view. In addition, their potential use in medicine as highly selective toxins is being explored. In contrast, the evolutionary history of RIP encoding genes has remained traditionally underexplored. In recent years, accumulation of large genomic data has fueled research on this issue and revealed unexpected information about the origin and evolution of RIP toxins. In this review we summarize the current evidence available on the occurrence of different evolutionary mechanisms (gene duplication and losses, horizontal gene transfer, synthesis de novo and domain combination) involved in the evolution of the RIP gene family. Finally, we propose a revised nomenclature for RIP genes based on their evolutionary history.

A novel mutation of NFIX causes Sotos-like syndrome (Malan syndrome) complicated with thoracic aortic aneurysm and dissection

Malan syndrome has recently been characterized to present Sotos-like phenotypes, such as intellectual disability and macrocephaly, with mutations in the NFIX gene. Herein, we report a 38-year-old patient with a novel single adenine insertion mutation in exon 2 of the NFIX gene (c.290_291insA). He developed early-onset thoracic aortic aneurysm and dissection, which was a rare complication but deserves particular attention in relatively long-lived patients with Sotos-like phenotypes.

Using Clickable NAD+ Analogs to Label Substrate Proteins of PARPs.

ADP-ribosylation has been well known as an important posttranslational modification, which is catalyzed by a family of enzymes called poly(ADP-ribose) polymerases (PARPs). PARPs transfer of a single or multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD+) to specific amino acids on substrate proteins. Through mono- or poly-ADP-ribosylation enzymatic activities, PARPs regulate various biological processes, including DNA damage repair, chromatin remodeling, transcriptional regulation, RNA processing and metabolism. Notably, PARP inhibitors are in clinical trials to treat human diseases, in particular cancer. To further investigate the biological function of PARPs, and to achieve better therapeutic effect of PARP inhibitors, it is important to identify the physiological substrates of PARPs. Here we describe a protocol to use clickable analog of nicotinamide adenine dinucleotide (NAD+) that can be applied for the detection, affinity purification and identification of substrate proteins of PARPs.

Development of a Genosensor for Sickle Cell Anemia Trait Determination

Sickle cell anemia (SCA) is a common recessive genetic condition in which patients produce hemoglobin S (HbS), an abnormal form of the protein, instead of hemoglobin A, the regular form. SCA is caused by the substitution of a single nitrogenous base adenine for thymine (GAG-> GTG), encoding valine instead of glutamine.1 Deoxygenation of HbS leads to its polymerization, damaging the cell membrane and reducing the lifetime of erythrocytes dramatically – which, in turn, leads to anemia. In some regions of Africa, up to 40% of the population possess its genetic trait, being considered a public health serious problem2. As the disease is possibly deadly, presents very low cure chances and has limited treatment, carriers determination is a key feature to assist in reproducibility decisions. Currently, trait determination is mainly performed by liquid chromatography, electrophoresis and polymerase chain reaction (PCR)3,4. These techniques are not widely used due to its complexity, lack of portability, high price and specialized personal and equipment demand. In this study we have developed an electrochemical genosensor for simple, low cost and rapid SCA carriers determination. Single DNA strands corresponding to the sequence of a SCA carrier were immobilized in gold platforms using the self-assembled-monolayers technique. The determination of SCA trait is then performed by analyzing the film’s charge transfer resistance using Electrochemical Impedance Spectroscopy. The genosensor was able to distinguish between healthy and unhealthy subjects with great precision besides displaying a wide linear range (0.01 to 7.5 µmol/L, R2 = 0.927). Furthermore, the device presented a detection limit of 7.0 nmol/L. We expect that such devices increases the number of SCA carriers determination, promoting early diagnosis and genetical counseling.

Analysis of mutations of MDR3 exons 9 and 23 in infants with parenteral nutrition-associated cholestasis.

The aim of this study was to investigate mutations of multidrug resistance 3 (MDR3) exons 9 and 23 in infants with parenteral nutrition-associated cholestasis (PNAC). A total of 41 infants with PNAC were enrolled in the study. Genomic DNA was extracted from the peripheral venous blood leukocytes of each patient and MDR3 exons 9 and 23 were amplified by polymerase chain reaction. One patient was identified who carried a frameshift mutation in MDR3 exon 23 (C.2793) that was caused by the insertion of a single adenine residue, while mutations were not found in MDR3 exon 23 in the other 40 patients. The clinical features of the patient with the MDR3 exon 23 frameshift mutation included high serum γ-glutamyl transferase levels, the absence of biliary dilatation and deformity in magnetic resonance cholangiopancreatography, and abnormal electrical capacitance tomography imaging of the liver. No mutations in MDR3 exon 9 were identified in any of the patients. All 41 PNAC patients recovered following oral ursodeoxycholic acid treatment. The C.2793 frameshift mutation in MDR3 exon 23 is potentially associated with the development of PNAC in infants.