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1.
Phys Chem Chem Phys ; 20(33): 21381-21389, 2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-30101268

RESUMO

Recent studies have evidenced that oxidatively damaged DNA, which potentially leads to carcinogenic mutations and aging, may result from the direct absorption of low-energy photons (>250 nm). Herein, the primary species, i.e., ejected electrons and base radicals associated with such damage in duplexes with an alternating guanine-cytosine sequence are quantified by nanosecond transient absorption spectroscopy. The one-photon ionization quantum yield at 266 nm is 1.2 × 10-3, which is similar to those reported previously for adenine-thymine duplexes. This means that the simple presence of guanine, the nucleobase with the lowest ionization potential, does not affect photo-ionization. The transient species detected after 3 µs are identified as deprotonated guanine radicals, which decay with a half-time of 2.5 ms. Spectral assignment is made with the help of quantum chemistry calculations (TD-DFT), which for the first time, provide reference absorption spectra for guanine radicals in duplexes. In addition, our computed spectra predict the changes in transient absorption expected for hole localization as well as deprotonation (to cytosine and bulk water) and hydration of the radical cation.


Assuntos
Citosina/efeitos da radiação , Radicais Livres/síntese química , Guanina/efeitos da radiação , Oligodesoxirribonucleotídeos/efeitos da radiação , Citosina/química , Dano ao DNA , Elétrons , Guanina/química , Meia-Vida , Modelos Químicos , Oligodesoxirribonucleotídeos/química , Fótons , Teoria Quântica , Raios Ultravioleta , Água/química
2.
Faraday Discuss ; 207(0): 181-197, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29372211

RESUMO

There 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.


Assuntos
Adenina/química , Adenina/efeitos da radiação , Timina/química , Timina/efeitos da radiação , Raios Ultravioleta , Radicais Livres/química , Radicais Livres/efeitos da radiação , Teoria Quântica
3.
J Phys Chem B ; 121(48): 10782-10792, 2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29116794

RESUMO

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.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Polietilenoimina/química , Polilisina/química , Estrutura Molecular , Espectrometria de Fluorescência , Fatores de Tempo
4.
Eur J Pharm Sci ; 103: 122-127, 2017 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-28330769

RESUMO

Electrostatic polymer-DNA complexes (polyplexes) have been widely investigated for DNA delivery, and remarkable differences in transfection efficacy have been seen among the materials. For example, polyethyleneimine (PEI) mediates DNA transfection more effectively than poly(l-lysine) (PLL). Biophysical properties of the polyplexes may explain their different properties in gene delivery. We investigated the structural dynamics in DNA polyplexes, especially the material exchange between the core and shell regions of the PEI and PLL polyplexes. Steady-state fluorescence spectroscopy and double labeling based fluorescence resonance energy transfer (FRET) techniques were used to study the DNA polyplexes. According to our results there is a clear difference between these two polymers: core exchange takes place in PEI but not in PLL polyplexes. Such differences in structural dynamics of polyplexes explain, at least partly, the differences in DNA release and transfection efficacy at cellular level.


Assuntos
DNA/química , Polietilenoimina/química , Polilisina/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Peso Molecular , Plasmídeos , Eletricidade Estática
5.
J Phys Chem Lett ; 7(19): 3949-3953, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27636653

RESUMO

Adenyl 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.


Assuntos
Adenina/química , DNA/química , Radicais Livres/química , Espectroscopia de Ressonância de Spin Eletrônica , Radical Hidroxila/química , Fótons , Teoria Quântica , Espectrofotometria , Raios Ultravioleta
6.
J Phys Chem Lett ; 7(11): 2020-3, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27163876

RESUMO

The reaction intermediate in the path leading to UV-induced formation of adenine dimers A═A and AA* is identified for the first time quantum mechanically, using PCM/TD-DFT calculations on (dA)2 (dA: 2'deoxyadenosine). In parallel, its fingerprint is detected in the absorption spectra recorded on the millisecond time-scale for the single strand (dA)20 (dA: 2'deoxyadenosine).


Assuntos
Adenina/química , Teoria Quântica , Dimerização , Raios Ultravioleta
7.
J Phys Chem B ; 117(36): 10405-13, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-23941196

RESUMO

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.


Assuntos
DNA/química , Polietilenoimina/química , Polilisina/química , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , DNA/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Tamanho da Partícula
8.
J Am Chem Soc ; 135(18): 6951-7, 2013 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-23570657

RESUMO

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(ß-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) × 10(4) M(-1). A binding constant in this range is necessary but not sufficient for effective transfection.


Assuntos
Antineoplásicos/farmacologia , Carbono/química , DNA/química , Técnicas de Transferência de Genes , Vetores Genéticos/farmacologia , Polímeros/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Polímeros/síntese química , Polímeros/química , Relação Estrutura-Atividade
9.
J Control Release ; 154(2): 171-6, 2011 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-21699928

RESUMO

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(ß-amino esters), cationic biodegradable polymers with DNA complexation and transfection capability. From the large library of poly(ß-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(ß-amino esters) than for 25 kDa polyethylenimines, yet poly(ß-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.


Assuntos
DNA/metabolismo , Polímeros/metabolismo , Transfecção/métodos , DNA/genética , Nanopartículas , Ligação Proteica/fisiologia , Espectrometria de Fluorescência/métodos , Fatores de Tempo
10.
J Phys Chem B ; 115(8): 1895-902, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21291220

RESUMO

Polyethylenimine (PEI) is a cationic DNA condensing polymer that facilitates gene transfer into the mammalian cells. The highest gene transfer with branched PEI is obtained at high nitrogen/phosphate (N/P) ratios with free PEI present. The small molecular weight PEI alone is not able to mediate DNA transfection. Here, we used recently developed time-resolved fluorescence spectroscopic method to study the mechanism of PEI-DNA complex formation and to investigate how free PEI, mean molecular weight, and branching of PEI affect the complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that for both linear and branched high-molecular-weight PEI the complexation takes place in two steps, but the small-molecular-weight branched PEI complexed DNA at a single step. According to the binding constants obtained from time-resolved spectroscopic measurements, the affinity of N/P complexation per nitrogen atom is highest for LPEI and weakest for BPEI, whereas SPEI-DNA complexation showed intermediate values. Thus, the binding constant alone does not give adequate measure for transfection efficiency. On the other hand, the presence of intermediate states during the polyplex formation seems to be favorable for the gene transfection. Free PEI had no impact on the physical state of PEI-DNA complexes, even though it was essential for gene transfection in the cell culture. In conclusion, the molecular size and topology of PEI have direct influence on the DNA complexation but the free PEI does not. Free PEI must facilitate transfection at the cellular level and not via indirect effects on the PEI-DNA complexes.


Assuntos
DNA/química , Polietilenoimina/química , Transfecção/métodos , Animais , Linhagem Celular , Cricetinae , Cricetulus , Haplorrinos , Coelhos , Espectrometria de Fluorescência , Fatores de Tempo
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