Excited State Pathways Leading to Formation of Adenine Dimers

International audienceThe reaction intermediate in the path leading to UV-induced formation ofadenine dimers A=A and AA* is identified for the first time quantum mechanically, usingPCM/TD-DFT calculations on (dA)2 (dA: 2′deoxyadenosine). In parallel, its fingerprint isdetected in the absorption spectra recorded on the millisecond time-scale for the singlestrand (dA)20 (dA: 2′deoxyadenosine)

Poly(β-amino ester)–DNA complexes: Time-resolved fluorescence and cellular transfection studies

A large number of different polymers have been developed and studied for application as DNA carriers for non-viral gene delivery, but the DNA binding properties are not understood. This study describes the efficiency of nanoparticle formation by time-resolved fluorescence measurements for poly(β-amino esters), cationic biodegradable polymers with DNA complexation and transfection capability. From the large library of poly(β-amino esters) ten polymers with different transfection efficacies were chosen for this study. The binding constants for nanoparticle formation were determined and compared to with the same method. Although the DNA binding efficiency of the amine groups are similar for both types of polymers, the overall binding constants are an order of magnitude smaller for poly(β-amino esters) than for 25 kDa polyethylenimines, yet poly(β-amino esters) show comparable DNA transfection efficacy with polyethylenimines. Within this series of polymers the transfection efficacy showed increasing trend in association with relative efficiency of nanoparticle formation.Academy of FinlandNational Institutes of Health (U.S.) (Grant CA132091)National Institutes of Health (U.S.) (Grant CA115527

Binding Affinity and Mechanism of Polymer-DNA Polyplexes for Gene Delivery

The mechanism of polyethylenimine−DNA, poly(L-lysine)−DNA, peptide–DNA, and PBAE–DNA complex formation was studied by a time-resolved spectroscopic method. The data were analysed by a cooperative model for multivalent ligand binding to multisubunit substrate. The formation of polyplexes with polyethylenimines, poly(L-lysine) and peptide (KK)₂KGGC is observed to be positively cooperative and negatively cooperative with PBAEs. Polymers with positive cooperativity reach about 100% saturation in binding DNA, whereas for polymers with negative cooperativity, the saturation level remains at about 80–90%. The type of amine groups (primary, secondary and tertiary) of the polymers has an effect on the binding constants and the degree of cooperativity. The effects of pH, type of amine groups and polymer structure on the mechanism of the polyplex formation were studied with polyethylenimines (PEI) and poly(L-lysine) (PLL). At pH 5.2 and 7.4 for PEIs and PLL, the formation of the polyplex core was observed to be complete at N/P = 2, at which point nearly all DNA phosphate groups were bound by polymer amine groups. At higher N/P ratios, excess polymer binds to the core polyplex, forming a shell over the core. At pH 9.2, the core is formed at higher N/P ratios than at lower pH levels except for PLL, which behaves similarly at all pH levels. The overall cooperative binding constants are higher at pH 5.2 than at 9.2 due to the higher degree of amine group protonation at lower pH levels. The ionic strength and pH affect the binding mechanism with peptide (KK)₂KGGC polyplexes, but changing the buffer does not. Molecular weight shows a clear effect on the mechanism and efficiency of the polyplex formation: for the high-molecular weight polymers (BPEI and PLL), the saturation level is reached at lower N/P ratios than for low-molecular weight polymers (SPEI and peptide). In the absence of excess PEI, the transgene expression levels are lower than in the presence of it. However, the fluorescence properties of the polyplexes in the absence and the presence of excess PEI are similar. Hence, the original structure of the polyplex core is retained during the shell formation. The molecular structures of the poly(β-amino ester)s (PBAEs) can be modified in a controlled way with the accuracy of single carbon unit. The effect of very small changes in the polymer structure on the formation of the polyplexes was studied by changing the length of the backbone and the side chain, by adding end caps to the polymers and by changing the molecular weight of the polymers. For PBAEs without end caps, the highest saturation levels and overall binding constants were observed for the linear backbones and side chains when the number of carbons was four or five, respectively. The end-capping of PBAEs increases the amine density and the efficiency of polyplex formation, which is observed as higher saturation levels for the end-capped PBAEs. The presence of an OH-group in the end cap induces a change in the binding mechanism. The length of the backbone and the side chain of PBAEs were observed to be important via amine density, hydrophobicity and steric hindrance to the complex formation. High-molecular weight PBAEs formed polyplexes more effectively than smaller ones

Adenine radicals generated in alternating AT duplexes by direct absorption of low-energy UV radiation

International audienceThere is increasing evidence that the direct absorption of photons with energies that are lower than the ionization potential of nucleobases may result in oxidative damage to DNA. The present work, which combines nanosecond transient absorption spectroscopy and quantum mechanical calculations, studies this process in alternating adenine–thymine duplexes (AT)n. We show that the one-photon ionization quantum yield of (AT)10 at 266 nm (4.66 eV) is (1.5 ± 0.3) × 10−3. According to our PCM/TD-DFT calculations carried out on model duplexes composed of two base pairs, (AT)1 and (TA)1, simultaneous base pairing and stacking does not induce important changes in the absorption spectra of the adenine radical cation and deprotonated radical. The adenine radicals, thus identified in the time-resolved spectra, disappear with a lifetime of 2.5 ms, giving rise to a reaction product that absorbs at 350 nm. In parallel, the fingerprint of reaction intermediates other than radicals, formed directly from singlet excited states and assigned to AT/TA dimers, is detected at shorter wavelengths. PCM/TD-DFT calculations are carried out to map the pathways leading to such species and to characterize their absorption spectra; we find that, in addition to the path leading to the well-known TA* photoproduct, an AT photo-dimerization path may be operative in duplexes

UV-Induced Adenine Radicals Induced in DNA A-Tracts: Spectral and Dynamical Characterization

International audienceAdenyl radicals generated in DNA single and double strands, (dA)20 and (dA)20·(dT)20, by one- and two-photon ionization by 266 nm laser pulses decay at 600 nm with half-times of 1.0 ± 0.1 and 4 ± 1 ms, respectively. Though ionization initially forms the cation radical, the radicals detected for (dA)20 are quantitatively identified as N6-deprotonated adenyl radicals by their absorption spectrum, which is computed quantum mechanically employing TD-DFT. Theoretical calculations show that deprotonation of the cation radical induces only weak spectral changes, in line with the spectra of the adenyl radical cation and the deprotonated radical trapped in low temperature glasses

Time-Resolved Fluorescence Spectroscopy Reveals Fine Structure and Dynamics of Poly(l‑lysine) and Polyethylenimine Based DNA Polyplexes

Structural dynamics of the polyethylenimine–DNA and poly­(l-lysine)–DNA complexes (polyplexes) was studied by steady-state and time-resolved fluorescence spectroscopy using the fluorescence resonance energy transfer (FRET) technique. During the formation of the DNA polyplexes, the negative phosphate groups (P) of DNA are bound by the positive amine groups (N) of the polymer. At N/P ratio 2, nearly all of the DNA’s P groups are bound by the polymer N groups: these complexes form the core of the polyplexes. The excess polymer, added to this system to increase the N/P ratio to the values giving efficient gene delivery, forms a positively charged shell around the core polyplex. We investigated whether the exchange between the core and shell regions of PEI and PLL polyplexes takes place. Our results demonstrated a clear difference between the two studied polymers. Shell PEI can replace PEIs previously attached to DNA in the polyplex core, while PLL cannot. Such a dynamic structure of PEI polyplexes compared to a more static one found for PLL polyplexes partially explains the observed difference in the DNA transfection efficiency of these polyplexes. Moreover, the time-resolved fluorescence spectroscopy revealed additional details on the structure of PLL polyplexes: in between the core and shell, there is an intermediate layer where both core and shell PLLs or their parts overlap

The Effect and Role of Carbon Atoms in Poly(β-amino ester)s for DNA Binding and Gene Delivery

Polymeric vectors for gene delivery are a promising alternative for clinical applications, as they are generally safer than viral counterparts. Our objective was to further our mechanistic understanding of polymer structure–function relationships to allow the rational design of new biomaterials. Utilizing poly­(β-amino ester)­s (PBAEs), we investigated polymer–DNA binding by systematically varying the polymer molecular weight, adding single carbons to the backbone and side chain of the monomers that constitute the polymers, and varying the type of polymer end group. We then sought to correlate how PBAE binding affects the polyplex diameter and ζ potential, the transfection efficacy, and its associated cytotoxicity in human breast and brain cancer cells in vitro. Among other trends, we observed in both cell lines that the PBAE–DNA binding constant is biphasic with the transfection efficacy and that the optimal values of the binding constant with respect to the transfection efficacy are in the range (1–6) × 10⁴ M^–1. A binding constant in this range is necessary but not sufficient for effective transfection

Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(l‑lysine)–DNA Polyplexes

The mechanism of polyethylenimine–DNA and poly­(l-lysine)–DNA complex formation at pH 5.2 and 7.4 was studied by a time-resolved spectroscopic method. The formation of a polyplex core was observed to be complete at approximately N/P = 2, at which point nearly all DNA phosphate groups were bound by polymer amine groups. The data were analyzed further both by an independent binding model and by a cooperative model for multivalent ligand binding to multisubunit substrate. At pH 5.2, the polyplex formation was cooperative at all N/P ratios, whereas for pH 7.4 at N/P < 0.6 the polyplex formation followed independent binding changing to cooperative binding at higher N/Ps