Wireframe DNA Origami for the Cellular Delivery of Platinum(II)-Based Drugs.

The DNA origami method has revolutionized the field of DNA nanotechnology since its introduction. These nanostructures, with their customizable shape and size, addressability, nontoxicity, and capacity to carry bioactive molecules, are promising vehicles for therapeutic delivery. Different approaches have been developed for manipulating and folding DNA origami, resulting in compact lattice-based and wireframe designs. Platinum-based complexes, such as cisplatin and phenanthriplatin, have gained attention for their potential in cancer and antiviral treatments. Phenanthriplatin, in particular, has shown significant antitumor properties by binding to DNA at a single site and inhibiting transcription. The present work aims to study wireframe DNA origami nanostructures as possible carriers for platinum compounds in cancer therapy, employing both cisplatin and phenanthriplatin as model compounds. This research explores the assembly, platinum loading capacity, stability, and modulation of cytotoxicity in cancer cell lines. The findings indicate that nanomolar quantities of the ball-like origami nanostructure, obtained in the presence of phenanthriplatin and therefore loaded with that specific drug, reduced cell viability in MCF-7 (cisplatin-resistant breast adenocarcinoma cell line) to 33%, while being ineffective on the other tested cancer cell lines. The overall results provide valuable insights into using wireframe DNA origami as a highly stable possible carrier of Pt species for very long time-release purposes.

Predicting the Three-Dimensional Structure of the c-KIT Proto-Oncogene Promoter and the Dynamics of Its Strongly Coupled Guanine Quadruplexes.

Guanine quadruplexes (G4s) play essential protective and regulatory roles within cells, influencing gene expression. In several gene-promoter regions, multiple G4-forming sequences are in close proximity and may form three-dimensional arrangements. We analyze the interplay among the three neighboring G4s in the c-KIT proto-oncogene promoter (WK1, WSP, and WK2). We highlight that the three G4s are structurally linked and their cross-talk favors the formation of a parallel structure for WSP. Relying on all-atom molecular dynamic simulations exceeding the µs time scale and using enhanced sampling methods, we provide the first computationally resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that can ultimately be used as a starting point for drug discovery.

Interplay of Three G-Quadruplex Units in the KIT Promoter.

The proto-oncogene KIT encodes for a tyrosine kinase receptor, which is a clinically validated target for treating gastrointestinal stromal tumors. The KIT promoter contains a G-rich domain within a relatively long sequence potentially able to form three adjacent G-quadruplex (G4) units, namely, K2, SP, and K1. These G4 domains have been studied mainly as single quadruplex units derived from short truncated sequences and are currently considered promising targets for anticancer drugs, alternatively to the encoded protein. Nevertheless, the information reported so far does not contemplate the interplay between those neighboring G4s in the context of the whole promoter, possibly thwarting drug-discovery efforts. Here we report the structural and functional study of the KIT promoter core sequence, in both single- and double-stranded forms, which includes all three predicted G4 units. By preventing the formation of alternatively one or two G4 units and by combining biophysical techniques and biological assays, we show for the first time that these quadruplexes cannot be analyzed independently, but they are correlated to each other. Our data suggest that, while K2 and K1 G-rich sequences retain the ability to fold into parallel G4 motifs within a long sequence, the SP G-rich domain contributes to G4 structure only together with K2. Remarkably, we have found that, in the context of a dynamic equilibrium between the three G4 units, the G4 formed by K1 has the most significant influence on the structure stability and on the biological role of the whole promoter.

BtsCI and BseGI display sequence preference in the nucleotides flanking the recognition sequence.

Restriction enzymes are the bread and butter of Molecular Biology. Nonetheless, how restriction enzymes recognize and cleave their target is not always clear. When developing a method for the enzymatic production of oligonucleotides, we noticed that type II endonucleases BtsCI and BseGI, which recognize the sequence GGATGNN^, perform incomplete digestions of DNA hairpins, with the top strand nick not always occurring correctly. We tested the cutting of synthetic hairpins containing all possible combinations of dinucleotides following the recognition site and our results show that all sequences containing one adenine following GGATG were digested more efficiently. We further show that the same sequence preference is also observable in double stranded DNA at higher Mg2+ concentrations and even in optimal conditions. Kinetic results show that BtsCI has a noteworthy difference in the first-rate constants between different sequences and between the two catalytic domains. An increase in Mg2+ resulted in a drastic decrease in the catalytic activity of the top (sense) strand that wasn't always accompanied by a nick in the bottom strand (antisense).

Enzymatic Synthesis of Single-Stranded Clonal Pure Oligonucleotides.

Single-stranded oligonucleotides, or oligodeoxyribonucleotides (ODNs), are very important in several fields of science such as molecular biology, diagnostics, nanotechnology, and gene therapy. They are usually chemically synthesized. Here we describe an enzymatic method which enables us to synthesize pure oligonucleotides which can be up to several hundred long bases.

Histamine-functionalized copolymer micelles as a drug delivery system in 2D and 3D models of breast cancer.

Histamine functionalized block copolymers based on poly(allyl glycidyl ether)-b-poly(ethylene oxide) (PAGE-b-PEO) were prepared with different ratios of histamine and octyl or benzyl groups using UV-initiated thiol-ene click chemistry. At neutral pH, the histamine units are uncharged and hydrophobic, while in acidic environments, such as in the endosome, lysosomes, or extracellular sites of tumours, the histamine groups are positively charged and hydrophilic. pH responsible polymer drug delivery systems is a promising route to site specific delivery of drugs and offers the potential to avoid side effects of systemic treatment. Our detailed in vitro experiments of the efficacy of drug delivery and the intracellular localization characteristics of this library of NPs in 2D and 3D cultures of breast cancer revealed that the 50% histamine-modified polymer loaded with DOX exhibited rapid accumulation in the nucleus of free DOX within 2 h. Confocal studies showed enhanced mitochondrial localization and lysosomal escape when compared to controls. From these combined studies, it was shown that by accurately tuning the structure of the initial block copolymers, the resulting self-assembled NPs can be designed to exploit histamine as an endosomal escape trigger and the octyl/benzyl units give rise to a hydrophobic core resulting in highly efficacious drug delivery systems (DDS) with control over intracellular localization. Optimization and rational control of the intracellular localization of both DDS and the parent drug can give nanomedicines a substantial increase in efficacy and should be explored in future studies.

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA.

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3'-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.

Toward unimolecular micelles with tunable dimensions using hyperbranched dendritic-linear polymers.

A library of amphiphilic, hyperbranched dendritic-linear polymers (HBDLPs) are successfully synthesized, and evaluated as potential unimolecular micelles. Hyperbranched macroinitiators (HBMI), extended with poly(ethylene glycol) methacrylate (P(OEGMA)), are afforded via a combination of self-condensing vinyl (co)polymerization (SCV(C)P) and atom transfer radical polymerization (ATRP), providing a versatile two-step synthetic route. The HBDLP architecture and chain lengths are varied, and the effect on the nanoparticle (NP) stability and properties are evaluated. The HBDLPs form predominantly stable and spherical NPs, and the NP dimensions could be tailored by the HBDLP characteristics. The NPs formed are of high molecular weight, and their stability varies with the properties of the corresponding HBDLP. Too small dendritic segment, or too low degree of PEGylation, results to some extent in NP aggregation, while higher molecular weight HBDLPs, with a high amount of hydrophilic segments, appears to form discrete unimolecular micelles. The versatility of the platform is further demonstrated by the convenience of forming a HBDLP with a more complex, linear copolymer extension instead of P(OEGMA).

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

DNA interaction of CuII, NiII and ZnII functionalized salphen complexes: studies by linear dichroism, gel electrophoresis and PCR.

The interaction of salphen-type NiII, CuII and ZnII complexes with native DNA was investigated by exploiting linear dichroism experiments. The NiII complex behaves as a typical intercalator, binding strongly and stiffening and unwinding the DNA. The strength of the DNA interaction is slightly weaker for the copper complex and much weaker for the zinc complex. Plasmid-DNA gel electrophoresis experiments indicated that while CuII and ZnII complexes do not induce the unwinding of supercoiled DNA, the NiII complex has a nuclease activity without the addition of external agents. On the other hand, as shown in the PCR assays, we demonstrate that, at the used concentrations, only the CuII complex is able to inhibit the DNA amplification mediated by Taq DNA polymerase. In this paper we have also reported a detailed characterization of the three compounds including 2D-NMR and ESI-mass experiments and X-ray single crystal structure of the copper and nickel compounds.

DNA binding studies and cytotoxicity of a dinuclear PtII diazapyrenium-based metallo-supramolecular rectangular box.

The interaction with native DNA of a 2,7-diazapyrenium-based ligand 1 and its Pt(II) rectangular metallacycle 2 is explored through circular and linear dichroism and fluorescence spectroscopies. The metal-free ligand 1 binds through intercalation, with a binding constant of approximately 5×10(5) M(-1), whereas the metallacycle 2 binds and bends the DNA with a binding constant of 7×10(6) M(-1). PCR assays show that metallo-supramolecular box 2 interferes with DNA transactions in vitro whereas the intercalator 1 does not. The metallacycle is active against four human cancer cell lines, with IC(50) values ranging between 3.1 and 19.2 µM and shows similar levels of efficacy, but a different spectrum of activity, to cisplatin.

Conjugation of testosterone modifies the interaction of mono-functional cationic platinum(II) complexes with DNA, causing significant alterations to the DNA helix.

Previously a range of androgen conjugates with non-conventional platinum(II) complexes have been synthesised with the aim of enhancing cellular delivery, and which have shown increased cytotoxic activity compared with non-steroidal compounds (M. J. Hannon et al., Dalton Trans., 2010, DOI: 10.1039/c0dt00838a). To further study this, the complexes have been assessed for their ability to bind to and alter the structure of DNA. All platinum(II) complexes studied herein bind to model nucleo-bases and DNA, but to our surprise, testosterone-based complexes caused the DNA helix to undergo significant unwinding and bending, whereas non-steroidal control complexes caused minimal structural alterations. These effects are similar to those cisplatin induces on DNA structure despite the fact that these compounds produce a monofunctional lesion. This ability attributed to interactions between the DNA helix and bulky steroidal skeleton of testosterone, coupled with the enhanced cellular delivery induced by the steroid make the steroid approach an exciting way to explore non-conventional platinum drug delivery.