Marco Deiana

Photochromic switching of the DNA helicity induced by azobenzene derivatives

The photochromic properties of azobenzene, involving conformational changes occurring upon interaction with light, provide an excellent tool to establish new ways of selective regulation applied to biosystems. We report here on the binding of two water-soluble 4-(phenylazo)benzoic acid derivatives (Azo-2N and Azo-3N) with double stranded DNA and demonstrate that the photoisomerization of Azo-3N leads to changes in DNA structure. In particular, we show that stabilization and destabilization of the B-DNA secondary structure can be photochemically induced in situ by light. This photo-triggered process is fully reversible and could be an alternative pathway to control a broad range of biological processes. Moreover, we found that the bicationic Azo-3N exhibited a higher DNA-binding constant than the monocationic Azo-2N pointing out that the number of positive charges along the photosensitive polyamines chain plays a pivotal role in stabilizing the photochrome-DNA complex.

Interactions of Isophorone Derivatives with DNA: Spectroscopic Studies

Interactions of three new isophorone derivatives, Isoa Isob and Isoc with salmon testes DNA have been investigated using UV-Vis, fluorescence and circular dichroism spectroscopic methods. All the studied compounds interact with DNA through intercalative binding mode. The stoichiometry of the isophorone/DNA adducts was found to be 1:1. The fluorescence quenching data revealed a binding interaction with the base pairs of DNA. The CD data indicate that all the investigated isophorones induce DNA modifications.

Specific Recognition of G-Quadruplexes Over Duplex-DNA by a Macromolecular NIR Two-Photon Fluorescent Probe

The implication of guanine-rich DNA sequences in biologically important roles such as telomerase dysfunction and the regulation of gene expression has prompted the search for structure-specific G-quadruplex agents for targeted diagnostic and therapeutic applications. Herein, we report on a near-infrared (NIR) two-photon poly(cationic) anthracene-based macromolecule able to selectively target G-quadruplexes (G4s) over genomic double-stranded DNA. In particular, the striking changes in its linear and third-order nonlinear optical properties, combined with the emergence of a strong induced electronic circular dichroism (ECD) signal upon binding to canonical and noncanonical DNA secondary structures allowed for a highly specific detection of several different G4s. Furthermore, through a detailed computational analysis we bring compelling evidence that our probe intercalation within G4s is a thermodynamically favored event, and we fully rationalize the spectroscopic evolution resulting from this complexation event by providing a reasonable explanation regarding the origin of the peculiar ECD effect that accompanies it.

Effective control of the intrinsic DNA morphology by photosensitive polyamines

Non-viral vectors for gene therapy such as DNA-cationic probe complexes offer important bio-safety advantages over viral approaches, due to their reduced pathogenicity, immunogenicity and cytotoxicity. In the present study we examine two polycationic water-soluble azobenzene derivatives (bis-Azo-2N and bis-Azo-3N) containing different linear unsubstituted polyamine moieties and we demonstrate the ability of such photochromes to destabilize the intrinsic B-DNA secondary structure in a concentration-dependent manner. Furthermore, through a detailed series of biophysical experiments, varying the photochrome conformation, temperature, salt and DNA concentration, we provide a detailed insight into the azobenzene-DNA binding pathway (Ka: bis-Azo-2N(trans)-DNA = 5.3 ± 0.3 × 104 M-1, Ka: bis-Azo-2N(cis)-DNA = 2.6 ± 0.2 × 104 M-1, Ka: bis-Azo-3N(trans)-DNA = 7.1 ± 0.4 × 104 M-1 and Ka: bis-Azo-3N(cis)-DNA = 5.1 ± 0.4 × 104 M-1) establishing the versatility of such materials as promising candidates for use in non-viral gene delivery systems.

Two-Photon Macromolecular Probe Based on a Quadrupolar Anthracenyl Scaffold for Sensitive Recognition of Serum Proteins under Simulated Physiological Conditions

The binding interaction of a biocompatible water-soluble polycationic two-photon fluorophore (Ant-PIm) toward human serum albumin (HSA) was thoroughly investigated under simulated physiological conditions using a combination of steady-state, time-resolved, and two-photon excited fluorescence techniques. The emission properties of both Ant-PIm and the fluorescent amino acid residues in HSA undergo remarkable changes upon complexation allowing the thermodynamic profile associated with Ant-PIm–HSA complexation to be accurately established. The marked increase in Ant-PIm fluorescence intensity and quantum yield in the proteinous environment seems to be the outcome of the attenuation of radiationless decay pathways resulting from motional restriction imposed on the fluorophore. Fluorescence resonance energy transfer and site-marker competitive experiments provide conclusive evidence that the binding of Ant-PIm preferentially occurs within the subdomain IIA. The pronounced hypsochromic effect and increased fluorescence enhancement upon association with HSA, compared to that of bovine serum albumin (BSA) and other biological interferents, makes the polymeric Ant-PIm probe a valuable sensing agent in rather complex biological environments, allowing facile discrimination between the closely related HSA and BSA. Furthermore, the strong two-photon absorption (TPA) with a maximum located at 820 nm along with a TPA cross section σ2 > 800 GM, and the marked changes in the position and intensity of the band upon complexation definitely make Ant-PIm a promising probe for two-photon excited fluorescence-based discrimination of HSA from BSA.

Probing the binding mechanism of photoresponsive azobenzene polyamine derivatives with human serum albumin

Control over chemical and biochemical processes by agents sensitive either to internal or external stimuli has attracted much attention in recent years. In particular photosensitive polyamines have been recently used to photo-trigger the hybridization/melting of DNA as well as to modify its intrinsic morphology. These results prompted us to synthesize azobenzene-based polyamine derivatives and study their impact towards human serum albumin (HSA), the principal extracellular protein in plasma, which is highly responsible for the proper biological activity exerted by exogenous compounds. It turns out that to assess and understand the binding mechanism of relevant compounds towards the HSA active sites is a critical step for the design of biomolecules-targeted probes. Herein, we show that both the mono-substituted Azo-4N and bi-substituted bis-Azo-4N azobenzene derivatives bind the protein template with moderate affinity and the number of positive charges along the polyamine moiety plays a pivotal role in stabilizing the photochrome–HSA adduct. Changes taking place in the fluorescence intensity of the tryptophan residue enabled us to determine the Ksv and kq parameters which provide evidence for a quenching driven by a static mechanism. Both ΔH and ΔS of the binding process being negative indicates that the HSA–photochrome association is mainly stabilized by a combination of long-range interactions of ionic nature. The overlap between the donor (Trp-214) and acceptor (photochrome) spectra allowed to calculate the distance (r) and the rate (kET) of energy transfer. Investigation of the HSA structural components reveals that the azobenzene derivatives, in both their conformations, slightly affect the overall protein secondary structure and they do not change its native state. Direct comparison of our results achieved by using photosensitive polyamines with those previously reported for biogenic and analogous polyamines bound to HSA reveals that the azo motif does not enhance to a large degree the overall binding affinity of the photoswitches towards the globular protein and contributes only a little to affect its intrinsic morphology.

Reversible Photocontrol of DNA Melting by Visible-Light-Responsive F4-Coordinated Azobenzene Compounds

Spatiotemporal control over the regulation of intra- and intermolecular motions in naturally occurring systems is systematically studied to expand the toolbox of mechanical operations in multicomponent nanoarchitectures. DNA is ideally suited for programming light-powered processes that are based on a minimalist molecular design. Here, the noncovalent incorporation of bistable photoswitches into B-like DNA moieties is shown to trigger the thermal transition midpoint of the duplexes by converting visible light into directed mechanical work by orchestrating the collective actions of the photoresponsive chromophores and the host DNA nanostructures. Besides its practical applications, the resulting hybrid nanosystem bears unique features of modulability, biocompatibility, reversibility, and addressability, which are key components for developing molecular photon-controlled programmed materials.

Correction: Interactions of Isophorone Derivatives with DNA: Spectroscopic Studies

The following information is missing from the Funding section: Wroclaw Centre of Biotechnology, Programme, The Leading National Research Centre (KNOW) provided funding for the publication of the results of the study.