RESUMO
Decades of crystallographic and NMR studies have produced canonical structural models of short DNA. However, no experimental method so far has been able to test these models in vivo, where DNA is long and constrained by interactions with membranes, proteins, and other molecules. Here, we employ high-resolution frequency-modulation AFM to image single long poly(dA)-poly(dT), poly(dG)-poly(dC), and lambda DNA molecules interacting with an underlying substrate that emulates the effect of biological constraints on molecular structure. We find systematic sequence-dependent variations in groove dimensions, indicating that the structure of DNA subject to realistic interactions may differ profoundly from canonical models. These findings highlight the value of AFM as a unique, single molecule characterization tool.
Assuntos
Bacteriófago lambda/química , DNA de Forma B/química , DNA Viral/química , Modelos Moleculares , Conformação de Ácido Nucleico , Poli dA-dT/químicaRESUMO
Purine radical cations (dAâ¢+ and dGâ¢+) are the primary hole carriers of DNA hole migration due to their favorable oxidation potential. Much less is known about the reactivity of higher energy pyrimidine radical cations. The thymidine radical cation (Tâ¢+) was produced at a defined position in DNA from a photochemical precursor for the first time. Tâ¢+ initiates hole transfer to dGGG triplets in DNA. Hole localization in a dGGG sequence accounts for â¼26% of Tâ¢+ formed under aerobic conditions in 9. Reduction to yield thymidine is also quantified. 5-Formyl-2'-deoxyuridine is formed in low yield in DNA when Tâ¢+ is independently generated. This is inconsistent with mechanistic proposals concerning product formation from electron transfer in poly(dA-T) sequences, following hole injection by a photoexcited anthraquinone. Additional evidence that is inconsistent with the original mechanism was obtained using hole injection by a photoexcited anthraquinone in DNA. Instead of requiring the intermediacy of Tâ¢+, the strand damage patterns observed in those studies, in which thymidine is oxidized, are reproduced by independent generation of 2'-deoxyadenosin- N6-yl radical (dAâ¢). Tandem lesion formation by dA⢠provides the basis for an alternative mechanism for thymidine oxidation ascribed to hole migration in poly(dA-T) sequences. Overall, these experiments indicate that the final products formed following DNA hole transfer in poly(dA-T) sequences do not result from deprotonation or hydration of Tâ¢+, but rather from deprotonation of the more stable dAâ¢+, to form dAâ¢, which produces tandem lesions in which 5'-flanking thymidines are oxidized.
Assuntos
Poli dA-dT/química , Sequência de Bases , Dano ao DNA , Transporte de Elétrons , Análise de Sequência de DNARESUMO
Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.
Assuntos
Replicação do DNA , DNA Ribossômico/química , Nucleossomos/metabolismo , Poli dA-dT/química , Origem de Replicação , Motivos de Aminoácidos , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Instabilidade Cromossômica , Sítios Frágeis do Cromossomo , Fragilidade Cromossômica , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae , Schizosaccharomyces , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
In this study it was theoretically shown that discovered by us recently (Brovarets' et al., Frontiers in Chemistry, 2018, 6:8; doi: 10.3389/fchem.2018.00008) high-energetical, significantly non-planar (symmetry C1), short-lived wobbled conformers of the classical Watson-Crick зТ(WC), reverse Watson-Crick зТ(rWC), Hoogsteen зТ(Ð) and reverse Hoogsteen зТ(rÐ) DNA base pairs are the intermediates of their pairwise ÐâТ(WC)/ÐâТ(rWC) â ÐâТ(H)/ÐâТ(rH) conformational transformations. These transitions do not require for their realization the energy-consumable anisotropic rotation of the amino group of A around the exocyclic C6-N6 bond. They are controlled by the non-planar transition states with quasi-orthogonal geometry (symmetry C1) joined by the single intermolecular (Т)N3H···N6(Ð) H-bond (~4 kcalâmol-1). The Gibbs free energies of activation for these non-dissociative, dipole-active conformational transitions consist 7.33 and 7.81 kcalâmol-1, accordingly. Quantum-mechanical (QM) calculations in combination with Bader's quantum theory of "Atoms in Molecules" (QTAIM) have been performed at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of QM theory in the continuum with ε = 4 under normal conditions.
Assuntos
Pareamento de Bases , Poli dA-dT/química , Termodinâmica , DNA/química , Ligação de Hidrogênio , Estrutura Molecular , Teoria QuânticaRESUMO
Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.
Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/química , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Cromatina/química , Cromatina/genética , DNA Fúngico/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Genoma Fúngico , Histonas/química , Histonas/genética , Poli dA-dT/química , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The ground- and excited-state interactions of Pt(ii) meso-tetrakis(4-N-methylpyridyl)porphyrin (PtTMPyP4) with polynucleotides [poly(dG-dC)]2 and [poly(dA-dT)]2 have been investigated using UV/visible, circular dichroism, and steady-state and time-resolved emission spectroscopy. PtTMPyP4 intercalates into [poly(dG-dC)]2 with Kâ¼ 10(6) M(-1). When bound to [poly(dG-dC)]2 in aerated solution there is a six-fold emission enhancement with 18 nm red-shift in emission maximum. Emission lifetimes are biexponential. In the presence of [poly(dA-dT)]2 at least two distinct groove-binding modes are observed, depending on the binding ratio. In [poly(dA-dT)]2 the emission intensity increases by a maximum factor of 17 with no shift in the emission spectrum. Three exponentials were required for lifetime fitting. The lower extent of emission enhancement in the presence of [poly(dG-dC)]2 suggests that a slow electron transfer may take place to guanine, which is significantly less efficient than that previously observed for PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP). The results are compared to those previously recorded with free base H2TMPyP4.
Assuntos
Metaloporfirinas/química , Compostos Organoplatínicos/química , Poli dA-dT/química , Polidesoxirribonucleotídeos/química , Dicroísmo Circular , DNA/química , DNA/metabolismo , Guanosina Monofosfato/química , Teoria Quântica , Espectrofotometria UltravioletaRESUMO
Doxorubicin and nogalamycin are antitumor antibiotics that interact with DNA via intercalation and threading mechanisms, respectively. Because the importance of water, particularly its impact on entropy changes, has been established in other biological processes, we investigated the role of water in these two drug-DNA binding events. We used the osmotic stress method to calculate the number of water molecules exchanged (Δnwater), and isothermal titration calorimetry to measure Kbinding, ΔH, and ΔS for two synthetic DNAs, poly(dA·dT) and poly(dG·dC), and calf thymus DNA (CT DNA). For nogalamycin, Δnwater<0 for CT DNA and poly(dG·dC). For doxorubicin, Δnwater>0 for CT DNA and Δnwater<0 for poly(dG·dC). For poly(dA·dT), Δnwater~0 with both drugs. Net enthalpy changes were always negative, but net entropy changes depended on the drug. The effect of water exchange on the overall sign of entropy change appears to be smaller than other contributions.
Assuntos
DNA/química , Doxorrubicina/química , Nogalamicina/química , Termodinâmica , Água/química , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Entropia , Poli dA-dT/química , Polidesoxirribonucleotídeos/químicaRESUMO
We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.
Assuntos
Campos Magnéticos , Técnicas Analíticas Microfluídicas/instrumentação , Genômica , Hidrodinâmica , Tamanho da Partícula , Poli dA-dT/química , RNA Mensageiro/isolamento & purificação , Análise de Célula Única/instrumentação , Fatores de TempoRESUMO
The role of microhydration in structural adjustments of the AT-tract in B-DNA was studied at the B97-D/def2-SV(P) level. The (dA:dT)5 complexes with 10 water molecules in minor and 15 water molecules in major grooves were studied. The obtained network of hydrogen bonds revealed the dependence between the groove width and the types of water patterns. In the minor groove, the following patterns were observed: interstrand one-water bridges similar to that of the Dickerson "water spine" and interstrand two-water bridges. The network of structural waters in the major groove is more diverse than that in the minor groove, which agrees with crystallographic data. As the major groove is wider, it is enriched by water molecules forming two- and three-water bridges. Results suggest the nucleobase-water interactions in both grooves prevent AT-tract twisting and its "collapse" along the minor groove. Whereby, a helix structure with narrow minor and wide major grooves is formed. The structural waters affect the polynucleotide conformation so that it becomes similar to poly(dA)·poly(dT) in fibers and acquires features of the A-tracts in DNA in solution. We suggest that formation of specific water patterns in both grooves is the factor responsible for stabilization of A-tracts with a narrowed minor groove, leading in turn to their strong intrinsic bending in DNA.
Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Poli dA-dT/química , Água/química , DNA de Forma B/química , Ligação de HidrogênioRESUMO
BACKGROUND: Homopolymeric tracts, particularly poly dA.dT, are enriched within the intergenic sequences of eukaryotic genomes where they appear to act as intrinsic regulators of nucleosome positioning. A previous study of the incomplete genome of the human malarial parasite Plasmodium falciparum reports a higher than expected enrichment of poly dA.dT tracts, far above that anticipated even in this highly AT rich genome. Here we report an analysis of the relative frequency, length and spatial arrangement of homopolymer tracts for the complete P. falciparum genome, extending this analysis to twelve additional genomes of Apicomplexan parasites important to human and animal health. In addition, using nucleosome-positioning data available for P. falciparum, we explore the correlation of poly dA.dT tracts with nucleosome-positioning data over key expression landmarks within intergenic regions. RESULTS: We describe three apparent lineage-specific patterns of homopolymeric tract organization within the intergenic regions of these Apicomplexan parasites. Moreover, a striking pattern of enrichment of overly long poly dA.dT tracts in the intergenic regions of Plasmodium spp. uniquely extends into protein coding sequences. There is a conserved spatial arrangement of poly dA.dT immediately flanking open reading frames and over predicted core promoter sites. These key landmarks are all relatively depleted in nucleosomes in P. falciparum, as would be expected for poly dA.dT acting as nucleosome exclusion sequences. CONCLUSIONS: Previous comparative studies of homopolymer tract organization emphasize evolutionary diversity; this is the first report of such an analysis within a single phylum. Our data provide insights into the evolution of homopolymeric tracts and the selective pressures at play in their maintenance and expansion.
Assuntos
Malária/parasitologia , Plasmodium falciparum/genética , DNA Intergênico/genética , DNA Intergênico/metabolismo , Expressão Gênica , Genoma de Protozoário , Humanos , Malária/patologia , Nucleossomos/metabolismo , Fases de Leitura Aberta/genética , Plasmodium falciparum/metabolismo , Poli dA-dT/química , Poli dA-dT/metabolismo , Regiões não Traduzidas/genéticaRESUMO
Understanding unwinding and melting of double helical DNA is very important to characterize role of DNA in replication, transcription, translation etc. Sequence dependent melting thermodynamics is used extensively for detecting promoter regions but melting studies are generally done for short oligonucleotides. This study reports several molecular dynamics (MD) simulations of homopolymeric poly(dA).poly(dT) as regular oligonucleotide fragments as well as its corresponding polymeric constructs with water and charge-neutralizing counterions at different temperatures ranging from 300 to 400 K. We have eliminated the end-effect or terminal peeling propensity by employing MD simulation of DNA oligonucleotides in such a manner that gives rise to properties of polymeric DNA of infinite length. The dynamic properties such as basepairing and stacking geometry, groove width, backbone conformational parameters, bending, distribution of counter ions and number of hydrogen bonds of oligomeric and polymeric constructs of poly(dA).poly(dT) have been analyzed. The oligomer shows terminal fraying or peeling effect at temperatures above 340 K. The polymer shows partial melting at elevated temperatures although complete denaturations of basepairs do not take place. The analysis of cross strand hydrogen bonds shows that the number of N-H···O hydrogen bonds increases with increase in temperature while C-H···O hydrogen bond frequencies decrease with temperature. Restructuring of counterions in the minor groove with temperature appear as initiation of melting in duplex structures.
Assuntos
DNA/química , Poli dA-dT/química , Polímeros/química , Termodinâmica , Pareamento de Bases , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Oligonucleotídeos/química , Temperatura , Água/químicaRESUMO
Doxorubicin (DOX) is an important anthracycline antibiotic whose intricate features of binding to DNAs, not yet fully understood, have been the object of intense debate. The dimerization equilibrium has been studied at pH = 7.0, I = 2.5 mM, and T = 25 °C. A thermodynamic and kinetic study of the binding of doxorubicin to DNA, carried out by circular dichroism, viscometry, differential scanning calorimetry, fluorescence, isothermal titration calorimetry, and T-jump relaxation measurements, has enabled us to characterize for the first time two different types of calf thymus DNA (ctDNA)/DOX complexes: PD1 for C(DOX)/C(DNA) < 0.3, and PD2 for higher drug content. The nature of the PD1 complex is described better in light of the affinity of DOX with the synthetic copolymers [poly(dA-dT)]2 and [poly(dG-dC)]2. The formation of PD1 has been categorized kinetically as a two-step mechanism in which the fast step is the groove binding in the AT region, and the slow step is the intercalation into the GC region. This bifunctional nature provides a plausible explanation for the high PD1 constant obtained (K1 = 2.3 × 10(8) M(-1)). Moreover, the formation of an external aggregate complex ctDNA/DOX (PD2) at the expense of PD1, with K2 = 9.3 × 10(5) M(-1), has been evinced.
Assuntos
Adutos de DNA/química , DNA/química , Doxorrubicina/química , Animais , Bovinos , DNA/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Poli dA-dT/química , Poli dA-dT/metabolismo , Polidesoxirribonucleotídeos/química , Polidesoxirribonucleotídeos/metabolismo , Termodinâmica , Temperatura de Transição , ViscosidadeRESUMO
Deoxyribonucleic acid (DNA) is a vital molecule for life since it contains genetic information. However, DNA has recently been reported to have unique properties that make it suitable for bionanoelectronic applications, such as the possibility of electrical conductivity and self-organisation. Self-assembled DNA network structures have been observed on several substrates, but the detailed self-assembly mechanism has yet to be determined. The present study investigates self-assembled structures of DNA both theoretically and experimentally. We developed a reaction-diffusion model and used it to investigate pattern formations observed by atomic force microscopy. The computational results qualitatively replicate the network patterns of DNA molecules based on a quantitative agreement with the surface size and timescale. The model can account for the effect of the DNA concentration on pattern formation. Furthermore, peculiar geometric patterns are simulated for mica and highly oriented pyrolytic graphite surfaces.
Assuntos
Silicatos de Alumínio/química , Grafite/química , Modelos Químicos , Poli dA-dT/química , DNA/química , Difusão , Microscopia de Força AtômicaRESUMO
Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant scenarios that are impacted by DNA deformability.
Assuntos
Sequência Rica em At/genética , DNA/química , Sequências Repetidas Invertidas/genética , Modelos Moleculares , Nucleossomos/química , Poli dA-dT/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Composição de Bases , Simulação por Computador , DNA/metabolismo , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Poli dA-dT/metabolismo , TermodinâmicaRESUMO
This study has investigated the formation patterns resulting from the self-assembly of deoxyribonucleic acid (DNA) on highly oriented pyrolytic graphite (HOPG), using both experimental and molecular dynamics approaches. Under optimized conditions based on pretreatment of HOPG surface and specific solution concentrations, DNA is found to self-assemble to form various patterned networks. The associated self-assembly mechanism is elucidated using coarse-grained molecular dynamics simulations and fractal dimension analysis. The results of this work demonstrate an effective technique allowing the formation of arrays of negatively charged biomacromolecules on negatively charged HOPG surfaces.
Assuntos
DNA/síntese química , Grafite/química , Simulação de Dinâmica Molecular , Poli dA-dT/química , DNA/química , Microscopia de Força AtômicaRESUMO
Dicationic 2,6-diphenylpyrazines, aza-analogues and prodrugs were synthesized; evaluated for DNA affinity, activity against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) in vitro, efficacy in T. b. r. STIB900 acute and T. b. brucei GVR35 CNS mouse models. Most diamidines gave poly(dA-dT)2 ΔTm values greater than pentamidine, IC50 values: T. b. r. (4.8-37nM) and P. f. (10-52nM). Most diamidines and prodrugs gave cures for STIB900 model (11, 19a and 24b 4/4 cures); 12 3/4 cures for GVR35 model. Metabolic stability half-life values for O-methylamidoxime prodrugs did not correlate with STIB900 results.
Assuntos
Antiprotozoários/síntese química , Compostos Aza/química , Pentamidina/química , Pró-Fármacos/síntese química , Pirazinas/química , Animais , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Cátions/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos , Mioblastos/citologia , Testes de Sensibilidade Parasitária , Pentamidina/uso terapêutico , Pentamidina/toxicidade , Plasmodium falciparum/efeitos dos fármacos , Poli dA-dT/química , Poli dA-dT/metabolismo , Pró-Fármacos/uso terapêutico , Pró-Fármacos/toxicidade , Ratos , Relação Estrutura-Atividade , Temperatura de Transição , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológicoRESUMO
The interaction of mitoxantrone with alternating Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT) duplex has been studied by absorption, fluorescence and Circular Dichroism (CD) spectroscopy at Drug to Phosphate base pair ratios D/P=20.0-0.04. Binding to GC polymer occurs in two distinct modes: partial stacking characterized by red shifts of 18-23nm at D/P=0.2-0.8 and external binding at D/P=1.0-20.0 whereas that to AT polymer occurs externally in the entire range of D/P. The binding constant and number of binding sites is 3.7×10(5)M(-1), 0.3 and 1.3× 10(4)M(-1), 1.5 in GC and AT polymers, respectively at low D/P ratios. CD binding isotherms show breakpoints at D/P=0.1, 0.5 and 0.25, 0.5 in GC and AT polymers, respectively. The intrinsic CD bands indicate that the distortions in GC polymer are significantly higher than that in AT polymer. Docking studies show partial insertion of mitoxantrone rings between to GC base pairs in alternating GC polymer. Side chains of mitoxantrone interact specifically with base pairs and DNA backbone. The studies are relevant to the understanding of suppression or inhibition of DNA cleavage on formation of ternary complex with topoisomerase-II enzyme and hence the anti cancer action.
Assuntos
Antineoplásicos/metabolismo , Mitoxantrona/metabolismo , Poli dA-dT/metabolismo , Polidesoxirribonucleotídeos/metabolismo , Análise Espectral , Absorção , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Poli dA-dT/química , Polidesoxirribonucleotídeos/químicaRESUMO
The spermine conjugate of the cationic porphyrin ligand (1) selectively induced the B-Z transition of the [(dA-T)n]2 sequence at low salt concentrations. The [(dG-C)n]2 sequence was not transformed into the Z-form. The B-Z transition was induced via an intermediate DNA conformation, which was formed by the external binding and formation of an assembly of 1 onto B-DNA.
Assuntos
DNA de Forma B/química , DNA Forma Z/química , Poli dA-dT/química , Porfirinas/química , Espermina/química , Dicroísmo Circular , Colorimetria , Conformação de Ácido Nucleico , Polidesoxirribonucleotídeos/químicaRESUMO
The transfection of DNA in gene therapy largely depends on the possibility of obtaining its condensation. The details of nanoparticle formation are essential for functioning, as mediated by the diverse elements containing molecular structure, ionic strength in mediums, and condensing motivator. Here, we report two kinds of DNA condensing agents based on simple cyclic/rigid polyamine molecules, having evaluated their structural effect on nanoparticle formation. The reversible condensation-dissociation process was achieved by ion-switching, attributing to a possible condensing mechanism-competitive building of external hydrogen bonds. Using poly[(dA-dT)2] and poly[(dG-dC)2] as substrates, respectively, circular dichroism (CD) signals clearly presented dissimilar interactions between polyamines and both rich sequences, implying potential preference for G-C sequence. The presence of divalent ion Zn(2+) as an efficient motivator accelerated the achievement of DNA condensation, and an accessible schematic model was depicted to explain the promotion in detail. In addition, by comparison with the behaviors of linear polyamines, differences between condensation and aggregation were explicitly elucidated in aspects of morphology and surface charges, as well as induced condition. The present work may have the potential to reveal the precise mechanism of DNA nanoparticle formation and, in particular, be applied to gene delivery as an efficient nonviral vector.
Assuntos
DNA Circular/química , DNA/química , Plasmídeos/química , Poliaminas/química , Animais , Bovinos , Ligação de Hidrogênio , Nanopartículas/química , Conformação de Ácido Nucleico , Poli dA-dT/química , Polidesoxirribonucleotídeos/química , Zinco/químicaRESUMO
Targeted delivery of antigens to dendritic cells (DCs) is a promising vaccination strategy. However, to ensure immunity, the approach depends on coadministration of an adjuvant. Here we ask whether targeting of both adjuvant and antigen to DCs is sufficient to induce immunity. Using a protein ligation method, we develop a general approach for linking the immune stimulant, poly dA:dT (pdA:dT), to a monoclonal antibody (mAb) specific for DEC205 (DEC). We show that DEC-specific mAbs deliver pdA:dT to DCs for the efficient production of type I interferon in human monocyte-derived DCs and in mice. Notably, adaptive T-cell immunity is elicited when mAbs specific for DEC-pdA:dT are used as the activation stimuli and are administered together with a DC-targeted antigen. Collectively, our studies indicate that DCs can integrate innate and adaptive immunity in vivo and suggest that dual delivery of antigen and adjuvant to DCs might be an efficient approach to vaccine development.