Immunostimulatory CpG oligonucleotides form defined three-dimensional structures: results from an NMR study

The DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' is known to stimulate the innate immune system of vertebrae. The immunostimulatory activity is based on the activation of Toll-like receptor 9 (TLR9). While it is known that the CG dinucleotide of the eicosamer has to be unmethylated, the structural basis of the recognition of the DNA through the receptor remains unclear. Oligodeoxynucleotides containing the sequence of the eicosamer, or a portion thereof, ranging in length from hexamer to pentaeicosamer were studied by (1)H NMR spectroscopy. Based on two-dimensional NMR spectra, a number of resonances could be unambiguously assigned. For all oligonucleotides, structural transitions were detected upon heating, as monitored by the line width and chemical shift of low-field resonances. This includes the TC dinucleotide of the 5'-terminal portion, which does not have any clear base-pairing partners. The melting transitions, together with the NOESY cross-peaks, demonstrate that structure formation occurs well beyond the core hexamer 5'-GACGTT-3', a fact that may be important for understanding the molecular recognition by the Toll-like receptors of the innate immune system.     

Modification of Magnetite Nanoparticles with Triazine-Based Dendrons and Their Application as Drug-Transporting Systems

The following research aims at the synthesis of magnetite nanoparticles functionalized with triazine-based dendrons and the application of the obtained materials as effective sorptive materials dedicated to acidic bioactive compounds. The adopted synthetic approach involved: (1) the synthesis of nanosized Fe₃O₄ particles via classic co-precipitation method, (2) the introduction of amine groups on their surface leading to materials’ precursor, and (3) the final synthesis of branched triazine-based dendrons on the support surface by an iterative reaction between cyanuric chloride (CC) and piperazine (p) or diethylenetriamine (DETA) via nucleophilic substitution. The characterized materials were tested for their adsorptive properties towards folic acid, 18β–glycyrrhetinic acid, and vancomycin, showing high adsorption capacities varying in the ranges of 53.33–401.61, 75.82–223.71, and 68.17–132.45 mg g⁻¹, respectively. The formed material–drug complexes were also characterized for the drug-delivery potential, performed as in vitro release studies at pH 2.0 and 7.4, which mimics the physiological conditions. The release profiles showed that the proposed materials are able to deliver up to 95.2% of the drugs within 48 h, which makes them efficient candidates for further biomedical applications.     

Recent Advances of DNA Tetrahedra for Therapeutic Delivery and Biosensing

The chemical and self-assembly properties of nucleic acids make them ideal for the construction of discrete structures and stimuli-responsive devices for a diverse array of applications. Amongst the various three-dimensional assemblies, DNA tetrahedra are of particular interest, as these structures have been shown to be readily taken up by the cell, by the process of caveolin-mediated endocytosis, without the need for transfection agents. Moreover, these structures can be readily modified with a diverse range of pendant groups to confer greater functionality. This minireview highlights recent advances related to applications of this interesting DNA structure including the delivery of therapeutic agents ranging from small molecules to oligonucleotides in addition to its use for sensing and imaging various species within the cell.     

Organometallic DNA–B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins

The synthesis and structural characterization of Co-(dN)₂₅-Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)₃₉-Cbl, which are organometallic DNA–B₁₂ conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA–Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA–Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA–Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)₂₅-Cbl, or radiolabeled vitamin B₁₂ (⁵⁷Co-CNCbl) and Co-(dN)₂₅-Cbl or Co-(dN)₃₉-Cbl. Binding of the new DNA–Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA–Cbl, Co-(dN)₁₈-Cbl. The contrasting affinities of TC versus IF and HC for the DNA–Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA–Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA–Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.     

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.     

Improvement of SNAr Reaction Rate by an Electron‐Withdrawing Group in the Crosslinking of DNA Cytosine‐5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor

DNA cytosine 5‐methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron‐deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism‐based modified nucleosides, 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) containing other nucleoside analogues, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐3‐cyano‐4‐halopyridine‐C‐nucleosides (d^XP^CN) and ODNs containing d^XP^CN, as more reactive inhibitors of DNMTs. Nucleophilic aromatic substitution (S_NAr) of the designed nucleosides, d^XP^CN, was faster than that of d^XP, and the ODN containing d^XP^CN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron‐withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.     

Silver Nanoparticles Modified by Carbosilane Dendrons and PEG as Delivery Vectors of Small Interfering RNA

The fact that cancer is one of the leading causes of death requires researchers to create new systems of effective treatment for malignant tumors. One promising area is genetic therapy that uses small interfering RNA (siRNA). These molecules are capable of blocking mutant proteins in cells, but require specific systems that will deliver RNA to target cells and successfully release them into the cytoplasm. Dendronized and PEGylated silver nanoparticles as potential vectors for proapoptotic siRNA (siMCL-1) were used here. Using the methods of one-dimensional gel electrophoresis, the zeta potential, dynamic light scattering, and circular dichroism, stable siRNA and AgNP complexes were obtained. Data gathered using multicolor flow cytometry showed that AgNPs are able to deliver (up to 90%) siRNAs efficiently to some types of tumor cells, depending on the degree of PEGylation. Analysis of cell death showed that complexes of some AgNP variations with siMCL-1 lead to ~70% cell death in the populations that uptake these complexes due to apoptosis.     

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.     

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.     

Adaptive Synthesis of Functional Amphiphilic Dendrons as a Novel Approach to Artificial Supramolecular Objects

Supramolecular structures, such as micelles, liposomes, polymerosomes or dendrimerosomes, are widely studied and used as drug delivery systems. The behavior of amphiphilic building blocks strongly depends on their spatial distribution and shape of polar and nonpolar component. This report is focused on the development of new versatile synthetic protocols for amphiphilic carbosilane dendrons (amp-CS-DDNs) capable of self-assembly to regular micelles and other supramolecular objects. The presented strategy enables the fine modification of amphiphilic structure in several ways and also enables the facile connection of a desired functionality. DLS experiments demonstrated correlations between structural parameters of amp-CS-DDNs and the size of formed nanoparticles. For detailed information about the organization and spatial distribution of amp-CS-DDNs assemblies, computer simulation models were studied by using molecular dynamics in explicit water.     

Glucose Conjugation of Anti‐HIV‐1 Oligonucleotides Containing Unmethylated CpG Motifs Reduces Their Immunostimulatory Activity

Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate–ODN conjugates have been reported to improve the cell‐specific delivery of ODNs through receptor mediated endocytosis. We tested the anti‐HIV activity and biochemical properties of the 5′‐end glucose‐conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV‐1 RNA in cell‐based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti‐HIV‐1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA–RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV‐1 dimerisation.     

Host-Assisted Delivery of a Model Drug to Genomic DNA: Key Information from Ultrafast Spectroscopy and in silico Study

Drug delivery to a target without adverse effects is one of the major criteria for clinical use. Herein, we have made an attempt to explore the delivery efficacy of SDS surfactant in a monomer and micellar stage during the delivery of the model drug, Toluidine Blue (TB) from the micellar cavity to DNA. Molecular recognition of pre-micellar SDS encapsulated TB with DNA occurs at a rate constant of k₁ ∼652 s⁻¹. However, no significant release of encapsulated TB at micellar concentration was observed within the experimental time frame. This originated from the higher binding affinity of TB towards the nano-cavity of SDS at micellar concentration which does not allow the delivery of TB from the nano-cavity of SDS micelles to DNA. Thus, molecular recognition controls the extent of DNA recognition by TB which in turn modulates the rate of delivery of TB from SDS in a concentration-dependent manner.     

Site-selective DNA hydrolysis by Ce(IV)-EDTA with the use of one oligonucleotide additive bearing two monophosphates

Two deoxyuridine derivatives each bearing a monophosphate group at the 5-position with a C3 linker, were incorporated into an oligonucleotide. By using this modified oligonucleotide, a bulge was formed at a predetermined position in a DNA substrate, and two monophosphate groups were placed at both junctions of the bulge. Upon treatment of the mixture with Ce(IV)-EDTA at pH 7.0, the phosphodiester linkages at the bulge site were selectively and efficiently hydrolyzed. The monophosphate groups introduced into the bulge site greatly accelerated site-selective DNA scission. Compared with the previously reported two-additive system, which combines two oligonucleotide additives each with a monophosphate at their termini, the present one-additive system is simpler and more convenient. Furthermore, site-selective DNA hydrolysis by using this one-additive system is successful even at high reaction temperatures (e.g., 55 degrees C). This reflects the thermodynamic stability of the duplexes formed between the substrate and the additive DNA.     

DNA Nanobarrel-Based Drug Delivery for Paclitaxel and Doxorubicin

Co-delivery of anticancer drugs and target agents by endogenous materials is an inevitable approach towards targeted and synergistic therapy. Employing DNA base pair complementarities, DNA nanotechnology exploits a unique nanostructuring method and has demonstrated its capacity for nanoscale positioning and templated assembly. Moreover, the water solubility, biocompatibility, and modifiability render DNA structure suitable candidate for drug delivery applications. We here report single-stranded DNA tail conjugated antitumor drug paclitaxel (PTX), and the co-delivery of PTX, doxorubicin and targeting agent mucin 1 (MUC-1) aptamer on a DNA nanobarrel carrier. We investigated the effect of tail lengths on drug release efficiencies and dual drug codelivery-enabled cytotoxicity. Owing to the rapidly developing field of structural DNA nanotechnology, functional DNA-based drug delivery is promising to achieve clinical therapeutic applications.     

Live-Cell Sensing of Telomerase Activity by Using Hybridization-Sensitive Fluorescent Oligonucleotide Probes

Live-cell sensing of telomerase activity with simple and efficient strategies remains a challenging target. In this work, a strategy for telomerase sensing by using hybridization-sensitive fluorescent oligonucleotide probes is reported. In the presence of telomerase and dNTPs, the designed supporting strand was extended and generated the hairpin structure that catalyzed the next telomerase extending reaction. The special extension mechanism increased the local concentration of another supporting strand and telomerase, which resulted in enhanced telomerase activity. The hybridization-sensitive oligonucleotide probes bound to the hairpin catalyst and generated turn-on fluorescence. This method realized the sensing of telomerase activity in HeLa cell extract with a detection limit below 1.6×10⁻⁶ IU μL⁻¹. The real-time in situ observation of telomerase extension was achieved in living HeLa cells. This strategy has been applied to monitor the efficiency of telomerase-targeting anticancer drugs in situ.     

Optimization of triple-helix-directed DNA cleavage by benzoquinoquinoxaline-ethylenediaminetetraacetic acid conjugates

The formation of triple-helical structures of DNA is based on sequence-specific recognition of oligopyrimidine.oligopurine stretches of double-helical DNA. Triple-helical structures can be stabilized by DNA-binding ligands. Benzoquinoquinoxaline (BQQ) derivatives are among the most potent intercalating-type agents known to stabilize DNA triple-helical structures. We previously reported the conversion of BQQ into a triplex-directed DNA cleaving agent, namely BQQ-ethylenediaminetetraacetic acid (EDTA), by coupling of 6-(3-aminopropylamino)BQQ to a suitable ethylenediaminetetraacetic acid derivative, and we demonstrated the ability of this conjugate to cause double-stranded cleavage of DNA at the triplex site. However, this prototype derivative BQQ-EDTA conjugate showed lower affinity towards triplex DNA than BQQ itself. In the light of this observation, and guided by molecular modeling studies, we synthesized a second generation of BQQ-EDTA conjugates based on 6-[bis(2-aminoethyl)amino]- and 6-(3,3'-diamino-N-methyldipropylamino)-BQQ derivatives. We confirmed by DNA melting experiments that the new conjugates displayed an increased specific affinity towards triple helices when compared to the previously synthesized BQQ-EDTA. In addition, the efficiency of these new agents in triplex-specific binding and cleavage was demonstrated by triplex-directed double-stranded cleavage of plasmid DNA.