New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure.

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.

pilE G-Quadruplex Is Recognized and Preferentially Bound but Not Processed by the MutL Endonuclease from Neisseria gonorrhoeae Mismatch Repair Pathway.

The human pathogen Neisseria gonorrhoeae uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by N. gonorrhoeae mismatch repair (MMR) and a G-quadruplex (G4) located upstream of the pilE promoter. Using bioinformatics tools, we found a correlation between pilE variability and deletion of DNA regions encoding ngMutS or ngMutL proteins, the main participants in N. gonorrhoeae methyl-independent MMR. To understand whether the G4 structure could affect the ngMutL-mediated regulation of pilin antigenic variation, we designed several synthetic pilE G4-containing oligonucleotides, differing in length, and related DNA duplexes. Using CD measurements and biochemical approaches, we have showed that (i) ngMutL preferentially binds to pilE G4 compared to DNA duplex, although the latter is a cognate substrate for ngMutL endonuclease, (ii) protein binding affinity decreases with shortening of quadruplex-containing and duplex ligands, (iii) the G4 structure inhibits ngMutL-induced DNA nicking and modulates cleavage positions; the enzyme does not cleave DNA within G4, but is able to bypass this noncanonical structure. Thus, pilE G4 may regulate the efficiency of pilin antigenic variation by quadruplex binding to ngMutL and suppression of homologous recombination.

Crosstalk between G-Quadruplexes and Dnmt3a-Mediated Methylation of the c-MYC Oncogene Promoter.

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

Neisseria gonorrhoeae: DNA Repair Systems and Their Role in Pathogenesis.

Neisseria gonorrhoeae (a Gram-negative diplococcus) is a human pathogen and causative agent of gonorrhea, a sexually transmitted infection. The bacterium uses various approaches for adapting to environmental conditions and multiplying efficiently in the human body, such as regulation of expression of gene expression of surface proteins and lipooligosaccharides (e.g., expression of various forms of pilin). The systems of DNA repair play an important role in the bacterium ability to survive in the host body. This review describes DNA repair systems of N. gonorrhoeae and their role in the pathogenicity of this bacterium. A special attention is paid to the mismatch repair system (MMR) and functioning of the MutS and MutL proteins, as well as to the role of these proteins in regulation of the pilin antigenic variation of the N. gonorrhoeae pathogen.

Impact of G-Quadruplex Structures on Methylation of Model Substrates by DNA Methyltransferase Dnmt3a.

In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface.

Involvement of E. coli 6S RNA in Oxidative Stress Response.

6S RNA, a small non-coding RNA present in almost all bacteria, inhibits transcription via direct binding to RNA polymerase holoenzymes. The mechanism of 6S RNA action was investigated to a large extent in E. coli, however, lack of 6S RNA (ΔssrS) was demonstrated to be unfavorable but not essential for cell survival under various growth conditions. In the present study, we revealed, for the first time, a lethal phenotype of the ΔssrS strain in the presence of high concentrations of H2O2. This phenotype was rescued by complementation of the ssrS gene on a plasmid. We performed comparative qRT-PCR analyses on an enlarged set of mRNAs of genes associated with the oxidative stress response, allowing us to identify four genes known to be involved in this pathway (soxS, ahpC, sodA and tpx) that had decreased mRNA levels in the ΔssrS strain. Finally, we performed comparative proteomic analyses of the wild-type and ΔssrS strains, confirming that ΔssrS bacteria have reduced levels of the proteins AhpC and Tpx involved in H2O2 reduction. Our findings substantiate the crucial role of the riboregulator 6S RNA for bacterial coping with extreme stresses.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2'-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA-protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA-protein interactions because of high selectivity of cysteine trapping.

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure.

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase.

Type II restriction⁻modification (RM) systems are the most widespread bacterial antiviral defence mechanisms. DNA methyltransferase SsoII (M.SsoII) from a Type II RM system SsoII regulates transcription in its own RM system in addition to the methylation function. DNA with a so-called regulatory site inhibits the M.SsoII methylation activity. Using circular permutation assay, we show that M.SsoII monomer induces DNA bending of 31° at the methylation site and 46° at the regulatory site. In the M.SsoII dimer bound to the regulatory site, both protein subunits make equal contributions to the DNA bending, and both angles are in the same plane. Fluorescence of TAMRA, 2-aminopurine, and Trp was used to monitor conformational dynamics of DNA and M.SsoII under pre-steady-state conditions by stopped-flow technique. Kinetic data indicate that M.SsoII prefers the regulatory site to the methylation site at the step of initial protein⁻DNA complex formation. Nevertheless, in the presence of S-adenosyl-l-methionine, the induced fit is accelerated in the M.SsoII complex with the methylation site, ensuring efficient formation of the catalytically competent complex. The presence of S-adenosyl-l-methionine and large amount of the methylation sites promote efficient DNA methylation by M.SsoII despite the inhibitory effect of the regulatory site.

Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site.

BACKGROUND: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. METHODS: The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. RESULTS AND CONCLUSIONS: The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. GENERAL SIGNIFICANCE: The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.

Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs--commonalities and differences.

Bacterial 6S RNAs bind to the housekeeping RNA polymerase (σ(A)-RNAP in Bacillus subtilis) to regulate transcription in a growth phase-dependent manner. B. subtilis expresses two 6S RNAs, 6S-1 and 6S-2 RNA, with different expression profiles. We show in vitro that 6S-2 RNA shares hallmark features with 6S-1 RNA: Both (1) are able to serve as templates for pRNA transcription; (2) bind with comparable affinity to σ(A)-RNAP; (3) are able to specifically inhibit transcription from DNA promoters, and (4) can form stable 6S RNA:pRNA hybrid structures that (5) abolish binding to σ(A)-RNAP. However, pRNAs of equal length dissociate faster from 6S-2 than 6S-1 RNA, owing to the higher A,U-content of 6S-2 pRNAs. This could have two mechanistic implications: (1) Short 6S-2 pRNAs (<10 nt) dissociate faster instead of being elongated to longer pRNAs, which could make it more difficult for 6S-2 RNA-stalled RNAP molecules to escape from the sequestration; and (2) relative to 6S-1 RNA, 6S-2 pRNAs of equal length will dissociate more rapidly from 6S-2 RNA after RNAP release, which could affect pRNA turnover or the kinetics of 6S-2 RNA binding to a new RNAP molecule. As 6S-2 pRNAs have not yet been detected in vivo, we considered that cellular RNAP release from 6S-2 RNA might occur via 6S-1 RNA displacing 6S-2 RNA from the enzyme, either in the absence of pRNA transcription or upon synthesis of very short 6S-2 pRNAs (∼ 5-mers, which would escape detection by deep sequencing). However, binding competition experiments argued against these possibilities.

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification.

In this work, the possibility of constructing a thermo-switchable enzyme according to the "molecular gate" strategy is demonstrated. The approach is based on the covalent attachment of oligodeoxyribonucleotides to cysteine residues of an enzyme adjacent to its active center to form a temporal barrier for enzyme-substrate complex formation. The activity of the modified enzyme that had been studied here-the restriction endonuclease SsoII (R.SsoII)-was diminished by a factor of 180 at 25 °Ð¡ that almost abolished the enzymatic activity when compared with the unmodified enzyme. However, heating of the modified enzyme to 45 °Ð¡ resulted in a 30-fold increase of activity. The activity of unmodified R.SsoII also increased on heating from 25 to 45 °; however, the difference did not exceed a factor of 3-4. The changes in enzymatic activity observed were shown to be reversible for both the unmodified and the modified R.SsoII. Variation of the length and the sequence of the attached oligodeoxyribonucleotides might allow greater modulation of the activity of DNA-protein conjugates.

Conserved G-Quadruplex-Forming Sequences in Mammalian TERT Promoters and Their Effect on Mutation Frequency.

Somatic mutations in the promoter region of the human telomerase reverse transcriptase (hTERT) gene have been identified in many types of cancer. The hTERT promoter is known to be enriched with sequences that enable the formation of G-quadruplex (G4) structures, whose presence is associated with elevated mutagenicity and genome instability. Here, we used a bioinformatics tool (QGRS mapper) to search for G4-forming sequences (G4 motifs) in the 1000 bp TERT promoter regions of 141 mammalian species belonging to 20 orders, 5 of which, including primates and predators, contain more than 10 species. Groups of conserved G4 motifs and single-nucleotide variants within these groups were discovered using a block alignment approach (based on the Nucleotide PanGenome explorer). It has been shown that: (i) G4 motifs are predominantly located in the region proximal to the transcription start site (up to 400 bp) and are over-represented on the non-coding strand of the TERT promoters, (ii) 11 to 22% of the G4 motifs found are evolutionarily conserved across the related organisms, and (iii) a statistically significant higher frequency of nucleotide substitutions in the conserved G4 motifs compared to the surrounding regions was confirmed only for the order Primates. These data support the assumption that G4s can interfere with the DNA repair process and affect the evolutionary adaptation of organisms and species.

Bioinformatics Analysis of Global Diversity in Meningococcal Vaccine Antigens over the Past 10 Years: Vaccine Efficacy Prognosis.

Neisseria meningitidis (N. meningitidis) serogroup B (MenB) is the leading cause of invasive meningococcal disease worldwide. The pathogen has a wide range of virulence factors, which are potential vaccine components. Studying the genetic variability of antigens within a population, especially their long-term persistence, is necessary to develop new vaccines and predict the effectiveness of existing ones. The multicomponent 4CMenB vaccine (Bexsero), used since 2014, contains three major genome-derived recombinant proteins: factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA). Here, we assessed the prevalence and sequence variations of these vaccine antigens in a panel of 5667 meningococcal isolates collected worldwide over the past 10 years and deposited in the PubMLST database. Using multiple amino acid sequence alignments and Random Forest Classifier machine learning methods, we estimated the potential strain coverage of fHbp and NHBA vaccine variants (51 and about 25%, respectively); the NadA antigen sequence was found in only 18% of MenB genomes analyzed, but cross-reactive variants were present in less than 1% of isolates. Based on our findings, we proposed various strategies to improve the 4CMenB vaccine and broaden the coverage of N. meningitidis strains.

G-Quadruplex Formed by the Promoter Region of the hTERT Gene: Structure-Driven Effects on DNA Mismatch Repair Functions.

G-quadruplexes (G4s) are a unique class of noncanonical DNAs that play a key role in cellular processes and neoplastic transformation. Herein, we focused on the promoter region of human TERT oncogene, whose product is responsible for the immortality of cancer cells. It has been shown by chemical probing and spectroscopic methods that synthetic 96-nt DNAs modeling the wild-type G-rich strand of the hTERT promoter and its variants with G>A point substitutions corresponding to somatic driver mutations fold into three stacked parallel G4s with sites of local G4 destabilization caused by G>A substitutions in the G4 motif. These models were used to elucidate how the hTERT multiG4 affects the binding affinity and functional responses of two key proteins, MutS and MutL, involved in the initial stage of DNA mismatch repair (MMR) in Escherichiacoli and Neisseriagonorrhoeae with different MMR mechanisms. We have shown for the first time that (i) point substitutions do not affect the effective binding of these proteins to the hTERT G4 structure, and (ii) the endonuclease activity of MutL from N. gonorrhoeae is significantly suppressed by the stable G4 scaffold. It is likely that some of the genomic instability associated with G4 may be related to the blockage of human intrinsic methyl-independent MMR attempting to operate near G4 structures.

Restriction endonuclease SsoII with photoregulated activity--a "molecular gate" approach.

A novel method for regulating the activity of homodimeric proteins--"molecular gate" approach--was proposed and its usefulness illustrated for the type II restriction endonuclease SsoII (R.SsoII) as a model. The "molecular gate" approach is based on the modification of R.SsoII with azobenzene derivatives, which allows regulating DNA binding and cleavage via illumination with light. R.SsoII variants with single cysteine residues introduced at selected positions were obtained and modified with maleimidoazobenzene derivatives. A twofold change in the enzymatic activity after illumination with light of wavelengths of 365 and 470 nm, respectively, was demonstrated when one or two molecules of azobenzene derivatives were attached to the R.SsoII at the entrance of or within the DNA-binding site.

In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis.

By differential high-throughput RNA sequencing (dRNA-seq) we have identified "product RNAs" (pRNAs) as short as 8-12 nucleotides that are synthesized by Bacillus subtilis RNA polymerase (RNAP) in vivo using the regulatory 6S-1 RNA as template. The dRNA-seq data were confirmed by in vitro transcription experiments and Northern blotting. In our libraries, we were unable to detect statistically meaningful numbers of reads potentially representing pRNAs derived from 6S-2 RNA. However, pRNAs could be synthesized in vitro from 6S-2 RNA as template by the B. subtilis σ(A) RNAP. 6S-1 pRNA levels are low during exponential, increase in stationary, and burst during outgrowth from stationary phase, demonstrating that pRNA synthesis is a conserved regulatory mechanism, but a more dynamic and fine-tuning process than previously thought. Most pRNAs have a length of 8-15 nt, very few up to 24 nt. The average length of pRNAs tended to increase from stationary to outgrowth conditions. Synthesis of pRNA is initiated at C40 of 6S-1 RNA and U41 of 6S-2 RNA, yielding pRNAs with a 5'-terminal G or A residue, respectively. A B. subtilis 6S-1 RNA mutant strain encoding a pRNA with a 5'-terminal A residue showed the same relative distribution of ~14-nt pRNAs between the different growth states, but generally displayed lower pRNA levels than the reference strain encoding wild-type 6S-1 RNA. A ~two-fold lower affinity of the C40U mutant 6S-1 RNA towards σ(A) RNAP may have contributed to this reduction in pRNA levels. We infer that 6S-1 pRNA synthesis, although evolutionarily optimized for initiation with a +1G residue, is not primarily regulated at the transcription initiation level via growth phase-dependent variations in the cellular GTP pool.

Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair.

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.

Northern Blot Detection of Tiny RNAs.

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA.

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is 'nicked' rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I-DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I's binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.