Duplex DNA Research Articles

Development of an Artificial Nucleic Acid Skeleton Allowing for Unnatural-Type Triplex DNA Formation with Duplex DNA Having a TA Inversion Site.

Triplex DNA formation has generated much interest as a genomic targeting tool that directly targets duplex DNA. However, fundamental limitations in the base pairs of target duplex DNA sequences that can form stable triplex DNA have limited the application. Recently, we have reported on the recognition of CG and 5mCG base pairs by artificial nucleic acid derivatives with a 2'-deoxynebularine skeleton. Therefore, we attempted to explore the basic skeleton that is important for the development of new artificial nucleic acids allowing for the recognition of TA base pairs. In this study, we focused on a benzimidazole skeleton and introduced a hydroxyl group to enable one-point hydrogen bonding. We have synthesized artificial nucleoside analogues with hydroxyl group on the benzimidazole and incorporated their amidite derivatives into triplex forming oligonucleotides (TFOs). The gel shift assay was performed to evaluate the triplex DNA formation ability of synthesized TFOs, and TFOs containing hydroxybenzimidazole were successfully recognized TA base pairs for all four different sequences. Moreover, compared to the results for the TFOs containing benzimidazole, which suggested hydrogen bonding formation at the hydroxyl group. Therefore, hydroxybenzimidazole would be an important artificial nucleic acid skeleton for TA base pair recognition.

Design, synthesis and characterization of tetra substituted 2,3-dihydrothiazole derivatives as DNA and BSA targeting agents: advantages of the visible-light-induced multicomponent approach.

This report describes the visible-light-induced one-pot multicomponent regioselective synthesis of a series of 5-aroyl-3-((arylidene)amino)-2-((arylidene)hydrazono)-4-methyl-2,3-dihydrothiazoles as DNA and BSA targeting agents. The multicomponent condensation of thiocarbohydrazide and aldehydes with α-bromo-1,3-diketones, generated in situ by the bromination of unsymmetrical 1,3-diketones with NBS using white LED light as an environmental friendly source in the presence of EtOAc solvent furnished the titled 2,3-dihydrothiazole derivatives in excellent yields. The exact regioisomeric structure was identified unambiguously by employing multinuclear 2D-NMR spectroscopy [1H-13C] HMBC; [1H-13C] HMQC and [1H-15N] HMBC. Furthermore, the binding characteristics of the synthesized 2,3-dihydrothiazole derivatives were assessed with double-stranded calf-thymus DNA duplex (ct-DNA) and bovine serum albumin (BSA). Initial screening of all the synthesized 2,3-dihydrothiazole derivatives using various in silico techniques including molecular reactivity analysis, Lipinski rule and molecular docking, concluded 5-(4'-chlorobenzoyl)-3-((4''-methoxybenzylidene)amino)-2-(4'''-methoxybenzylidene)hydrazono)-4-methyl-2,3-dihydrothiazole derivative 6a as the most suitable compound for studying binding interaction with DNA and BSA. Additionally, to illustrate the ex vivo binding mode of 6a with DNA and BSA, several spectroscopic techniques viz. UV-visible, circular dichroism (CD), steady-state fluorescence and competitive displacement assays were carried out.

Enzymatic synthesis of ligand-bearing oligonucleotides for the development of metal-responsive DNA materials.

Metal-mediated artificial base pairs are some of the most promising building blocks for constructing DNA-based supramolecules and functional materials. These base pairs are formed by coordination bonds between ligand-type nucleobases and a bridging metal ion and have been exploited to develop metal-responsive DNA materials and DNA-templated metal arrays. In this review, we provide an overview of methods for the enzymatic synthesis of DNA strands containing ligand-type artificial nucleotides that form metal-mediated base pairs. Conventionally, ligand-bearing DNA oligomers have been synthesized via solid-phase synthesis using a DNA synthesizer. In recent years, there has been growing interest in enzymatic methods as an alternative approach to synthesize ligand-bearing DNA oligomers, because enzymatic reactions proceed under mild conditions and do not require protecting groups. DNA polymerases are used to incorporate ligand-bearing unnatural nucleotides into DNA, and DNA ligases are used to connect artificial DNA oligomers to natural DNA fragments. Template-independent polymerases are also utilized to post-synthetically append ligand-bearing nucleotides to DNA oligomers. In addition, enzymatic replication of DNA duplexes containing metal-mediated base pairs has been intensively studied. Enzymatic methods facilitate the synthesis of DNA strands containing ligand-bearing nucleotides at both internal and terminal positions. Enzymatically synthesized ligand-bearing DNAs have been applied to metal-dependent self-assembly of DNA structures and the allosteric control of DNAzyme activity through metal-mediated base pairing. Therefore, the enzymatic synthesis of ligand-bearing oligonucleotides holds great potential in advancing the development of various metal-responsive DNA materials, such as molecular sensors and machines, providing a versatile tool for DNA supramolecular chemistry and nanotechnology.

Controlling the thermally-driven crystallization of DNA-coated nanoparticles with formamide.

DNA-coated nanoparticles, also known as programmable atom equivalents (PAEs), facilitate the construction of materials with nanoscopic precision. Thermal annealing plays a pivotal role by controlling DNA hybridization kinetics and thermodynamics, which ensures the formation of intended structures. While various design handles such as particle size, DNA design, and salt concentration influence the stability of the DNA duplexes linking PAEs in a lattice, their influence on the system's melting temperature (Tm) often follows complicated trends that make rational tuning of self-assembly challenging. In this work, the denaturant formamide is used to precisely tune the thermal response of PAEs. Our results reveal a clear and predictable trend in the PAEs' response to formamide, enabling rational control over the Tm of a diverse set of PAE systems. Unlike adjustments made through alterations to PAE design or solution parameters such as ionic strength, formamide achieves its temperature shift without impacting the kinetics of assembly. As a result, PAEs can be rapidly crystallized at ambient temperatures, producing superlattices with similar quality to PAE crystals assembled through standard protocols that use higher temperatures. This study therefore positions formamide as a useful tool for enhancing the synthesis of complex nanostructures under mild conditions.

Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach.

Incorporation of artificial 3rd base pairs (unnatural base pairs, UBPs) has emerged as a fundamental technique in pursuit of expanding the genetic alphabet. 2,6-Dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN), a potential unnatural base pair (UBP) developed by Romesberg and colleagues, has been shown to have remarkable capability for replication within DNA. Crystal structures of a Taq polymerase/double-stranded DNA (ds-DNA) complex containing a DS-DN pair in the 3' terminus showed a parallelly stacked geometry for the pre-insertion, and an intercalated geometry for the post-insertion structure. Unconventional orientations of DS-DN inside a DNA duplex have inspired scientists to investigate the conformational orientations and structural properties of UBP-incorporated DNA. In recent years, computational simulations have been used to investigate the geometry of DS-DN within the DNA duplex; nevertheless, unresolved questions persist owing to inconclusive findings. In this work, we investigate the structural and dynamical properties of DS and DN inside a ds-DNA strand in aqueous solution considering both short and long DNA templates using polarizable, and non-polarizable classical MD simulations. Flexible conformational change of UBP with major populations of Watson-Crick-Franklin (WCF) and three distinct non-Watson-Crick-Franklin (nWCFP1, nWCFP2, nWCFO) conformations through intra and inter-strand flipping have been observed. Our results suggest that a dynamical conformational change leads to the production of diffierent conformational distribution for the systems. Simulations with a short ds-DNA duplex suggest nWCF (P1 and O) as the predominant structures, whereas long ds-DNA duplex simulations indicate almost equal populations of WCF, nWCFP1, nWCFO. DS-DN in the terminal position is found to be more flexible with occasional mispairing and fraying. Overall, these results suggest flexibility and dynamical conformational change of the UBP as well as indicate varied conformational distribution irrespective of starting orientation of the UBP and length og DNA strand.

Iridium(III)-based minor groove binding complexes as DNA photocleavage agents.

Transition metal complexes containing the qtpy ligand (2':4,4'':4',4'''-quaterpyridyl) are known to be DNA intercalators or minor groove binders. In this study, new tricationic iridium(III) complexes of qtpy are reported. Both [Ir(bpy)2(qtpy)]3+1 and [Ir(phen)2(qtpy)]3+2 display good water solubility as chloride salts. The complexes possess high-energy excited states, which are quenched in the presence of duplex DNA and even by the mononucleotides guanosine monophosphate and adenosine monophosphate. Further studies reveal that although the complexes bind to quadruplex DNA, they display a preference for duplex structures, which are bound with an order of magnitude higher affinities than their isostructural dicationic RuII-analogues. Detailed molecular dynamics simulations confirm that the complexes are groove binders through the insertion of, predominantly, the qtpy ligand into the minor groove. Photoirradiation of 1 in the presence of plasmid DNA confirms that this class of complexes can function as synthetic photonucleases by cleaving DNA.

DYNAMICS OF COMPLEMENTARY BASE-PAIRS OF DREW-DICKERSON DODECAMER ON THE MILLIAND MICROSECOND TIME SCALE ACCORDING TO THE DATA OF NUCLEAR SPIN RELAXATION DISPERSION R1ρ OF IMINO PROTONS

Drew-Dickerson Dodecamer (DDD) is one of the well-known DNA duplexes. It has been sufficiently well studied by various structure determination methods and appears to be a standard for developing new experimental techniques and molecular mechanical force fields. For this reason, the information about the dynamics of DDD on different time scales is important for constructing more accurate structural-dynamic models and carrying out molecular dynamics calculations of long-term time trajectories that allow simulation of real processes. In this work, the information on the dynamics of DDD base-pairs on the milli- and microsecond time scale, currently missing from the literature, was obtained by means of imino protons’ nuclear spin relaxation (R1ρ) dispersion. It is shown for all the base-pairs, except for terminal and pre-terminal ones, that any dynamic processes in the range of R1ρ method are not observed up to 300 K, but proceed on a faster time scale. For the pre-terminal GC pair, a pronounced dependence of the relaxation R1ρ on the applied spin-lock field strength is observed, which allowed us to extract the information about the pair closing dynamics using a fast exchange model with highly skewed state populations. These data supplement the information obtained previously on the dynamics of DDD complementary base-pairs by exchange-based water magnetization transfer experiments and provide a more complete view of the processes observed in DDD in relation to base-pair opening-closing.

Contiguous Silver(I)-Mediated Base Pairs of Imidazophenanthroline and Canonical Nucleobases in DNA Duplexes: Formation of Classical Duplexes versus Homodimer Formation.

Metal-mediated base pairs represent a topical alternative to canonical hydrogen-bonded base pairs. In this context, the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was introduced as an artificial nucleobase in a glycol nucleic acid-based nucleoside analogue into a DNA oligonucleotide in a way that the oligonucleotide contains a central block of six contiguous P residues. The ability to engage in Ag+-mediated base pairing was evaluated with respect to the four canonical nucleosides in positions complementary to P. Highly stabilizing Ag+-mediated base pairs were formed with cytosine and guanine (i.e., P-Ag+-C and P-Ag+-G base pairs), whereas the analogous base pairs with thymine and adenine were much less stable and hence formed incompletely. Surprisingly, the intermediate formation of a homodimeric duplex of the P-containing oligonucleotide was observed in all cases, albeit to a different extent. The homodimer is composed of P-Ag+-P base pairs and 18 overhanging mismatched canonical nucleobases. It demonstrates the obstacles present when designing metal-mediated base pairs as metal complexation may take place irrespective of the surrounding natural base pairs. Homodimer formation was found to be particularly prominent when the designated metal-mediated base pairs are of low stability, suggesting that homodimers and regular duplexes are formed in a competing manner.

Investigating the Cytotoxicity of Ru(II) Polypyridyl Complexes by Changing the Electronic Structure of Salicylaldehyde Ligands.

A novel class of Ru(II)-based polypyridyl complexes with an auxiliary salicylaldehyde ligand [Ru(phen)2(X-Sal)]BF4 {X: H (1), 5-Cl (2), 5-Br (3), 3,5-Cl2 (4), 3,5-Br2 (5), 3-Br,5-Cl (6), 3,5-I2 (7), 5-NO2 (8), 5-Me (9), 4-Me (10), 4-OMe (11), and 4-DEA (12), has been synthesized and characterized by elemental analysis, FT-IR, and 1H/13C NMR spectroscopy. The molecular structure of 4, 6, 9, 10, and 11 was determined by single-crystal X-ray diffraction analysis which revealed structural similarities. DFT and TD-DFT calculations showed that they also possess similar electronic structures. Absorption/emission spectra were recorded for 2, 3, 10, and 11. All Ru-complexes, unlike the pure ligands and the complex lacking the salicylaldehyde component, displayed outstanding antiproliferative activity in the screening test (10 μM) against CCRF-CEM leukemia cells underlining the crucial role of the presence of the auxiliary ligand for the biological activity. The two most active derivatives, namely 7 and 10, were selected for continuous assays showing IC50 values in the submicromolar and micromolar range against drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells, respectively. These two compounds were investigated in silico for their potential binding to duplex DNA well-matched and mismatched base pairs, since they showed remarkable selectivity indexes (2.2 and 19.5 respectively) on PBMC cells.

UltraFast PhotoInduced double duplex DNA invasion into a 400-mer dsDNA target

BackgroundNatural DNA restriction enzymes bind duplex DNA with high affinity at multiple sites; however, for some of the artificial chemical-based restriction moieties, invasion of the double-strand for efficient cleavage is an obstacle. We have previously reported photo-induced double-duplex invasion (pDDI) using 3-cyanovinylcarbazole (K)-containing probes for both the target strands that photo-crosslink with pyrimidine bases in a sequence-specific manner on both the strands, stabilizing the opened double-strand for cleavage. The drawback of the pDDI was low efficiency due to inter-probe cross-linking, solved by the inclusion of 5-cyano-uridine at −1 position on the complimentary strand with respect to K in both probes. Although this led to reduced inter-probe cross-linking, the pDDI efficiency was still low. ResultsHere, we report that inter-probe cross-linking and intra-probe cross-linking of a single probe is also leading to reduced pDDI efficiency. We addressed this problem by designing DDI probes to inhibit both inter-probe and intra-probe cross-linking. ConclusionBased on the new design of pDDI probe with 5-cyano uridine led to a drastic increase in the efficiency of pDDI in (400-mer) double-stranded DNA with only 1 s of photo-irradiation.

Dynamic Gold Nanostructures Based on DNA Self Assembly.

The combination of DNA nanotechnology and Nano Gold (NG) plasmon has opened exciting possibilities for a new generation of functional plasmonic systems that exhibit tailored optical properties and find utility in various applications. In this review, the booming development of dynamic gold nanostructures are summarized, which are formed by DNA self-assembly using DNA-modified NG, DNA frameworks, and various driving forces. The utilization of bottom-up strategies enables precise control over the assembly of reversible and dynamic aggregations, nano-switcher structures, and robotic nanomachines capable of undergoing on-demand, reversible structural changes that profoundly impact their properties. Benefiting from the vast design possibilities, complete addressability, and sub-10nm resolution, DNA duplexes, tiles, single-stranded tiles and origami structures serve as excellent platforms for constructing diverse 3D reconfigurable plasmonic nanostructures with tailored optical properties. Leveraging the responsive nature of DNA interactions, the fabrication of dynamic assemblies of NG becomes readily achievable, and environmental stimulation can be harnessed as a driving force for the nanomotors. It is envisioned that intelligent DNA-assembled NG nanodevices will assume increasingly important roles in the realms of biological, biomedical, and nanomechanical studies, opening a new avenue toward exploration and innovation.

6‐Aryl‐Substituted Harmanium Derivatives as Light‐Up Probes for Fluorimetric DNA Detection and Staining of Eukaryotic Cells

AbstractNine 6‐aryl‐substituted harmanium derivatives were synthesized by Suzuki‐Miyaura coupling of 6‐bromoharmane with arylboronic acids and subsequent N2‐benzylation. Depending on the substitution pattern of the aryl group the emission maxima cover a range between λfl=445 and 529 nm with quantum yields of Φfl=<0.01–0.92. The interactions with duplex DNA were investigated by photometric and fluorimetric titrations as well as CD and LD spectroscopy. These ligands bind to DNA by intercalation with binding constants of Kb=1–3×104 M−1. It was demonstrated by fluorescence microscopy that the 2‐benzyl‐6‐(3‐methoxyphenyl)harmanium derivative operates as a fluorescent light‐up probe in the complex biological environment of eukaryotic cells, namely in NIH 3T3 mouse fibroblasts, as it stains the perinuclear cytoplasm and distinct substructures of the nucleus preferentially as compared with the rest of the nucleoplasm.

Rapid Colorimetric Determination of microRNA (miRNA) Using an Autocatalytic Entropy-Driven Circuit (AEDC)

A colorimetric and rapid signal amplified microRNA (miRNA) detection system based on autocatalytic entropy-driven circuit (AEDC) is reported. The target miRNA-21 (miR-21) was firstly hybridized with the sticky end of the triple-stranded hybrid complex (S, which consisting of a reporter strand (P), a byproduct strand (R) and an assistant strand (Q)), and P strand, which contained the G-quadruplex sequence that was released through the toehold-mediated strand displacement reaction. The G-quadruplex sequence interacted with hemin to form the peroxidase-mimicking DNAzymes and catalyzed the H2O2-mediated oxidation of colorless 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS2−) to the green ABTS•−. Meanwhile, fuel strand (F) hybridized with S to release the target miR-21 and the waste duplex DNA product F-Q was formed, which brings the two split mimic target DNA sequences into proximity to form the intact mimic target DNA sequence (analogue of miR-21). The obtained mimic target DNA as well as the released miR-21 can be cyclically used to trigger autocatalytic EDC reaction, leading to the rapidly enhanced formation of mimic target DNA with the concomitant generation of a higher colorimetric signal. Finally, a colorimetric and rapid miR-21 system based on the hairpin-free AEDC reaction was successfully fabricated. This system also has been applied for the miRNA determination in serum, exhibiting the potential application in the clinical diagnosis.

Synthesis and Characterization of Ruthenium-Paraphenylene-Cyclopentadienyl Full-Sandwich Complexes: Cytotoxic Activity against A549 Lung Cancer Cell Line and DNA Binding Properties.

Novel full-sandwich (η5-Cp)-Ru-paraphenylene complexes with the general formula [(η5-Cp)nRu(η6-L)](PF6)n where n = 1-3 and L = biphenyl, p-terphenyl and p-quaterphenyl, were synthesized and characterized by means of spectroscopic and analytical techniques. The structures of the complexes [(η5-Cp)Ru(η6-biphenyl)](PF6) (1), [(η5-Cp)Ru(η6-terphenyl)](PF6) (3) and [(η5-Cp)2Ru(η6-terphenyl)](PF6)2 (4) was determined by X-ray single crystal methods. The interaction of the complexes [(η5-Cp)Ru(η6-quaterphenyl)]Cl, (6)Cl, and [(η5-Cp)2Ru(η6-quaterphenyl)]Cl2, (7)Cl2, with the DNA duplex d(5'-CGCGAATTCGCG-3')2 was studied using NMR techniques. The results showed that both complexes interacted non-specifically with both the minor and major grooves of the helix. Specifically, (6)Cl exhibited partial binding through intercalation between the T7 and T8 bases of the sequence without disrupting the C-G and A-T hydrogen bonds. Fluorometric determination of the complexes' binding constants revealed a significant influence of the number of connected phenyl rings in the paraphenylene ligand (L) on the binding affinity of their complexes with the d(5'-CGCGAATTCGCG-3')2. The complexes (6)Cl and (7)Cl2 were found to be highly cytotoxic against the A549 lung cancer cell line, with complex (6) being more effective than (7) (IC50 for (6)Cl: 17.45 ± 2.1 μΜ, IC50 for (7)Cl2: 65.83 ± 1.8 μΜ) and with a selectivity index (SI) (SI for (6)Cl: 1.1 and SI for (7)Cl2: 4.8).

Mechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process.

DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.

Characterization of human XPD helicase activity with single-molecule magnetic tweezers

XPD helicase is a DNA-unwinding enzyme involved in DNA repair. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5′ to 3′ direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA unwinding and single-stranded DNA translocation activities of human XPD in isolation. In our experimental setup, hXPD exhibited low unwinding processivity of ∼14 bp and slow unwinding rate of ∼0.3 bp/s. Measurements of the ssDNA translocation activity demonstrated that hXPD translocated on ssDNA at a similar rate as unwinding, revealing that slow rate was an intrinsic property of the hXPD translocation. Individual unwinding and translocation events were composed of pauses and runs with a distribution of lengths and rates. Analysis of these events unveiled similar mean run lengths and rates for unwinding and translocation, indicating that the unwinding behavior was a direct reflection of the translocation activity. The analysis also revealed that hXPD spent similar time stalling and unwinding/translocating. The detailed basal activity of hXPD reported here provides a baseline for future studies on how hXPD activity is regulated by other TFIIH components.

A CRISPR/Cas12a-assisted bacteria quantification platform combined with magnetic covalent organic frameworks and hybridization chain reaction

The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans-cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.

Distinctive physical properties of DNA shared by RNA polymerase II gene promoters and 5'-flanking regions of tRNA genes.

Numerous noncoding (nc)RNAs have been identified. Similar to the transcription of protein-coding (mRNA) genes, long noncoding (lnc)RNA genes and most of micro (mi)RNA genes are transcribed by RNA polymerase II (Pol II). In the transcription of mRNA genes, core promoters play an indispensable role; they support the assembly of the preinitiation complex (PIC). However, the structural and/or physical properties of the core promoters of lncRNA and miRNA genes remain largely unexplored, in contrast with those of mRNA genes. Using the core promoters of human genes, we analyzed the repertoire and population ratios of residing core promoter elements (CPEs) and calculated the following five DNA physical properties (DPPs): duplex DNA free energy, base stacking energy, protein-induced deformability, rigidity and stabilizing energy of Z-DNA. Here, we show that their CPE and DPP profiles are similar to those of mRNA gene promoters. Importantly, the core promoters of these three classes of genes have two highly distinctive sites in their DPP profiles around the TSS and position -27. Similar characteristics in DPPs are also found in the 5'-flanking regions of tRNA genes, indicating their common essential roles in transcription initiation over the kingdom of RNA polymerases.

Self-Penetrating Oligonucleotide Derivatives: Features of Self-Assembly and Interactions with Serum and Intracellular Proteins.

Lipophilic oligonucleotide derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.

The bacterial replication origin BUS promotes nucleobase capture

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).