DNA Ternary Complexes Research Articles

Interaction of γ-Polyglutamic Acid/Polyethyleneimine/Plasmid DNA Ternary Complexes with Serum Components Plays a Crucial Role in Transfection in Mice.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.

RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.

DNA Topoisomerases: As target for anti-cancer drugs

Topoisomerase inhibitors are agents designed to interfere with the action of topoisomerase enzymes I and II. Topoisomerases are enzymes that control the changes in DNA tridimensional structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. DNA Topoisomerases control the conformational changes in DNA topology by breaking and resealing DNA strands during normal cellular growth, that’s why these are essential enzymes. A major class of anticancer drugs acts as inhibitors of DNA Topoisomerases. This paper gives a brief review on Topoisomerase targeting drugs which shows anticancer activities. The mechanism of action of these drugs by inhibiting type I and type II DNA Topoisomerases are discussed. DNA topoisomerases, especially type II A topoisomerases, are proved therapeutic targets of anticancer and antibacterial drugs. Clinically successful topoisomerase-targeting anticancer drugs act through topoisomerase poisoning, which leads to replication fork arrest and double-strand break formation. Unfortunately, this unique mode of action is associated with the development of secondary cancers and cardiotoxicity. Structures of topoisomerase–drug–DNA ternary complexes have revealed the exact binding sites and mechanisms of topoisomerase poisons. It may also be possible to design catalytic inhibitors of topoisomerases by targeting certain inactive conformations of these enzymes. Furthermore, identification of various new bacterial topoisomerase inhibitors and regulatory proteins may inspire the discovery of novel human topoisomerase inhibitors. Thus, topoisomerases remain as important therapeutic targets of anticancer agents.

Molecular Assembly of Triplex of Duplexes from Homothyminyl-Homocytosinyl Cγ(S/R)-Bimodal Peptide Nucleic Acids with dA8/dG6 and the Cell Permeability of Bimodal Peptide Nucleic Acids.

Peptide nucleic acids (PNAs) are analogues of DNA with a neutral acyclic polyamide backbone containing nucleobases attached through a t-amide link on repeating units of aminoethylglycine (aeg). They bind to complementary DNA or RNA in a sequence-specific manner to form duplexes with higher stablity than DNA:DNA and DNA:RNA hybrids. We have recently explored a new type of PNA termed bimodal PNA (bm-PNA) designed with two nucleobases per aeg repeating unit of PNA oligomer and attached at Cα or Cγ of each aeg unit through a spacer sidechain. We demonstrated that Cγ-bimodal PNA oligomers with mixed nucleobase sequences bind concurrently two different complementary DNAs, forming double duplexes, one from each t-amide and Cγ face, sharing a common PNA backbone. In such bm-PNA:DNA ternary complexes, the two duplexes show higher thermal stability than individual duplexes. Herein, we show that Cγ(S/R)-bimodal PNAs with homothymines (T8) on a t-amide face and homocytosine (C6) on a Cγ-face form a conjoined pentameric complex consisting of a triplex (bm-PNA-T8)2:dA8 and two duplexes of bm-PNA-C6:dG6. The pentameric complex [dG6:Cγ(S/R)-bm-PNA:dA8:Cγ(S/R)-bm-PNA:dG6] exhibits higher thermal stability than the individual triplex and duplex, with Cγ(S)-bm-PNA complexes being more stable than Cγ(R)-bm-PNA complexes. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-order assemblies with DNA and RNA. The Cγ(S/R)-bimodal PNAs are shown to enter MCF7 and NIH 3T3 cells and exhibit low toxicity to cells.

Direct binding of TFE\u03b1 opens DNA binding cleft of RNA polymerase

Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

Cγ(S/R)-Bimodal Peptide Nucleic Acids (Cγ-bm-PNA) Form Coupled Double Duplexes by Synchronous Binding to Two Complementary DNA Strands.

Peptide nucleic acids (PNAs) are linear equivalents of DNA with a neutral acyclic polyamide backbone that has nucleobases attached via tert-amide link on repeating units of aminoethylglycine. They bind complementary DNA or RNA with sequence specificity to form hybrids that are more stable than the corresponding DNA/RNA self-duplexes. A new type of PNA termed bimodal PNA [Cγ(S/R)-bm-PNA] is designed to have a second nucleobase attached via amide spacer to a side chain at Cγ on the repeating aeg units of PNA oligomer. Cγ-bimodal PNA oligomers that have two nucleobases per aeg unit are demonstrated to concurrently bind two different complementary DNAs, to form duplexes from both tert-amide side and Cγ side. In such PNA:DNA ternary complexes, the two duplexes share a common PNA backbone. The ternary DNA 1:Cγ(S/R)-bm-PNA:DNA 2 complexes exhibit better thermal stability than the isolated duplexes, and the Cγ(S)-bm-PNA duplexes are more stable than Cγ(R)-bm-PNA duplexes. Bimodal PNAs are first examples of PNA analogues that can form DNA2:PNA:DNA1 double duplexes via recognition through natural bases. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-level assemblies.

Discovery of New DNA Topoisomerase II Inhibitors using Structure Based Virtual Screening Method

DNA topoisomerases are proved therapeutic targets of anticancer and antibacterial drugs. Structures of topoisomerase–DNA and inhibitor ternary complexes have revealed the exact binding sites and mechanisms of topoisomerase poisons. There are two isoforms of Human Topoisomerase II; α and β. Both of them perform similar functions and their levels differ depending on the replicative activity and type of tissue. Topo IIα is preferentially expressed in proliferating cells. Thus selective Topo IIα inhibitors have been of particular interest in cancer therapy, as they may represent a more targeted approach to highly proliferative cells. In this study, we use structure based virtual screening method with molecules which are commercially available in the ZINC database. Docking studies were performed by Glide module available in Schrödinger software, Ligand filtration was also done to obtain an efficient collection of hit molecules by employing Lipinski “rule of five” and pharmacokinetic properties of the compounds were tested using Qikprop module. From approximately ten thousand compounds from Zinc database it was possible to select 4 top chemical structures with good inhibiting profile for topo II, with suitable ADME/Tox properties, thus comp. 1-4 could be the promising inhibitors of human topo IIα enzyme.

Reaction of Histone H1 with trans-Platinum Complexes and the Effect on DNA Platination.

Transplatin is an inactive platinum drug; however, a number of analogues, such as trans-EE and trans-PtTz, demonstrate promising antitumor activity in vitro and in vivo. Although the ultimate target is nuclear DNA, increasing evidence indicate that proteins also play important roles in the display of antitumor activity. The linker histone H1 is situated by the portal between the unwrapped DNA and the nucleosome core. Our recent study revealed that H1 can readily react with cisplatin, and the adducts tend to form ternary complexes with DNA. In this work, we have investigated the reaction of histone H1 with two antitumor-active trans-oriented complexes, trans-EE and trans-PtTz, and the effect of H1 upon the platination of DNA. The results show that trans-platinum drugs are much more reactive than cisplatin toward H1. Interestingly, in addition to the expected bidentate adducts (by displacement of the two labile chlorido ligands), also a tridentate adduct can be formed by displacement of one nonlabile carrier ligand of trans-EE or trans-PtTz. The trans-Pt/H1 adducts can then react with DNA and generate protein-Pt-DNA ternary complexes. Additionally, platinum can be transferred from trans-Pt/H1 adducts to DNA, generating binary trans-Pt/DNA complexes. Such a transfer of the platinum agent to DNA was not observed in the reaction of cisplatin. Furthermore, the detailed investigation carried out on a model peptide indicates that H1 promotes the DNA platination by trans- EE, while it reduces that of trans-PtTz and cisplatin. These results suggest that H1 can play a key role in the DNA platination and modulate the efficacy of different platinum agents.

Dual Role of φ29 DNA Polymerase Lys529 in Stabilisation of the DNA Priming-Terminus and the Terminal Protein-Priming Residue at the Polymerisation Site

Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3′ terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA polymerase, an enzyme that starts linear φ29 DNA replication using a terminal protein (TP) as primer. Our results show that single replacement of φ29 DNA polymerase residue Lys529 by Ala or Glu decreases the stabilisation of the primer-terminus at the polymerisation active site, impairing both the insertion of the incoming nucleotide when DNA and TP are used as primers and the translocation step required for the next incoming nucleotide incorporation. In addition, combination of the DNA polymerase mutants with a TP derivative at residue Glu233, neighbour to the priming residue Ser232, leads us to infer a direct contact between Lys529 and Glu233 for initiation of TP-DNA replication. Altogether, the results are compatible with a sequential binding of φ29 DNA polymerase residue Lys529 with TP and DNA during replication of TP-DNA.

Ternary Complexes with Core-Shell Bilayer for Double Level Targeted Gene Delivery: In Vitro and In Vivo Evaluation

Hyaluronic acid (HA)/polyethyleneimine-dexamethasone (PEI-Dex)/DNA ternary complexes with "core-shell" bilayer were developed for double level targeted gene delivery. A PEI1800-Dex, as a core, was applied to compact DNA into a nano-sized structure and facilitate the nuclear translocation of DNA after endocytosis into tumor cells, and a polyanion HA, as the outer corona, was employed to improve targeted tumor delivery and reduce cytotoxicity. PEI-Dex was synthesized and characterized by (1)H NMR. The characterizations of ternary complexes were investigated. Their biological properties, including transfection efficiency, cytotoxicity, cellular uptake and in vivo efficacy were evaluated systemically. Ternary complexes with the size of about 160nm exhibited the lowest cytotoxicity and the highest transfection efficiency in B16F10 cells among investigated complexes. The sub-cellular localization study confirmed that ternary complexes could facilitate more efficient cell uptake and nuclear transport of DNA than binary complexes. Moreover, Cy7-labeled ternary complexes obviously accumulated in the tumor after i.v. administration, indicating that ternary complexes could assist the DNA targeting to the tumor. In in vivo studies, HA/PEI1800-Dex/DNA ternary complexes showed confirmed anti-inflammation activity, and could significantly suppress tumor growth of tumor-bearing nude mice. HA/PEI-Dex/DNA ternary complexes might be a promising targeted gene delivery system.

Zinc-Chelated Poly(1-vinylimidazole) and a Carbohydrate Ligand Polycation Form DNA Ternary Complexes for Gene Delivery

Zinc-chelated poly(1-vinylimidazole) (PVIm-Zn) and a carbohydrate ligand polycation, a poly(l-lysine) conjugated with lactose molecules (PLL-Lac), have formed DNA ternary complexes for gene delivery. The particle size of the PVIm-Zn/DNA complexes with negative zeta potential was decreased by the addition of the PLL-Lac. The resulting PLL-Lac/PVIm-Zn/DNA ternary complexes, which exhibited the pH-dependent dissociation of the PLL-Lac, mediated more gene expression than the PVIm/DNA binary complexes. The PLL-Lac/PVIm-Zn/DNA complexes with the specific recognition of cell surface receptors mediated the highest gene expression without cytotoxicity at a relatively lower charge ratio (positive/negative = 2.5). These results suggest that the pH-dependent dissociation of the carbohydrate ligands after the recognition of cell surface receptors, including the physicochemical and biochemical function of PVIm-Zn, played an important role in gene expression.

Release of DNA binary complexes from the ternary complexes by carboxymethyl Poly(L-histidine)

The DNA ternary complexes with carboxymethyl poly(L-histidine) (CM-PLH) and poly(ethylenimine) (PEI) have released the DNA binary complexes with PEI by the protonation of CM-PLH at endosomal/lysosomal pH. The dissociation of the CM-PLH from the CM-PLH/PEI/DNA ternary complexes is proved by the fluorescence resonance energy transfer (FRET) analysis between the CM-PLH and PEI. The resulting PEI/DNA binary complexes easily released DNA, as compared with the CM-PLH/PEI/DNA ternary complexes, which was examined by competitive exchange with dextran sulfate. The release of the DNA binary complexes from the ternary complexes is promising mechanism for higher transfection activity by the CM-PLH/PEI/DNA ternary complexes.

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR–DNA complexes

The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

PH-Dependent Dissociation of Carbohydrate Ligand Polycations from DNA Ternary Complexes

Abstract Here, we have demonstrated that carbohydrate ligand polycations are pH-dependently dissociated from DNA polyion complexes. In response to pH, DNA ternary complexes have dissociated the ligand polycations, resulting in DNA binary complexes. Namely, the ternary complexes of DNA, a poly(l-histidine) modified with aminoethyl groups, PLH-NH2, and a poly(l-lysine) conjugated with lactose molecules, PLL-Lac, at pH 7.5 have dissociated the PLL-Lac polycations by the protonation of imidazole groups of PLH-NH2 at pH 6.0. The pH-dependent dissociation of the ligand polycations from DNA polyion complexes at endosomal pH is expected to offer unique design for DNA delivery systems.

Energy and structural analysis of double nucleic acid triplets

In order to investigate the energy and structural character of RNA–RNA triplets and RNA–DNA duplex base triplets, 64 sets of three-dimensional models of RNA–DNA duplex base triplets and mRNA–tRNA triplex base triplets were constructed and optimized by homologous modeling method using the software InsightII. The comparative statistical method and cluster analysis were adopted to study these features. The result showed: (i) all energy parameters of monomer RNA–DNA hybrid triplets and ternary complexes appeared significantly different; and some parameters related with overall molecules such as overall energy, bond energy and coulomb energy have statistically significant correlations between the structures in vacuum and aquatic solutions while other parameters, including theta energy, phi energy, hydrogen bond energy and non-bond energy, changed significantly, but not continuously. (ii) However, the case of mRNA–tRNA triplets was much more complicated in that only the bond energy's correlation coefficient is −0.8. Typically, the main contribution of GC pairs and G/A/U bases were interesting. The models of RNA–DNA hybrid triplets and mRNA–tRNA triplet should be helpful for the study of base pairing in codons and the biological effectiveness of antisense nucleic acids.

Association of CBP/p300 Acetylase and Thymine DNA Glycosylase Links DNA Repair and Transcription

DNA repair in chromatin is subject to topological constraints, suggesting a requirement for chromatin modification and remodeling activities. Thymine DNA glycosylase (TDG) initiates repair of G/T and G/U mismatches, commonly associated with CpG islands, by removing thymine and uracil moieties. We report that TDG associates with transcriptional coactivators CBP and p300 and that the resulting complexes are competent for both the excision step of repair and histone acetylation. Furthermore, TDG stimulates CBP transcriptional activity in transfected cells and reciprocally serves as a substrate for CBP/p300 acetylation. Remarkably, this acetylation triggers release of CBP from DNA ternary complexes and also regulates recruitment of repair endonuclease APE. These observations reveal a potential regulatory role for protein acetylation in base mismatch repair and a role for CBP/p300 in maintaining genomic stability.

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrine-copper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers. The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H2O2 and it preserves the capacity over a wide range of complex:DNA ratios. The rate of DNA clevage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when "free" copper ions are available, catecholamines may induce oxidative degradation of DNA and other biological macromolecules.