RESUMO
One-step functionalization of alginate with boronic acid groups allowed spontaneous formation of biocompatible hydrogels under basic conditions without additional complementary molecules or crosslinking agents. The dynamic nature of boronate ester bonds formed with vicinal diols present on alginate pyranose rings provided remarkable self-healing, injectable and multi-stimuli responsive properties to the material.
Assuntos
Alginatos/química , Ácidos Borônicos/química , Alginatos/síntese química , Alginatos/toxicidade , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Ácidos Borônicos/síntese química , Ácidos Borônicos/toxicidade , Frutose/química , Células HeLa , Humanos , Hidrogéis , ReologiaRESUMO
A rapid strategy for the covalent immobilization of DNA onto silicon-based materials using the UV-initiated radical thiol-ene reaction is presented in this study. Following this approach, thiol- and alkene-modified oligonucleotide probes were covalently attached in microarray format, reaching immobilization densities around 6 pmol·cm(-2). The developed methodology presents the advantages of spatially controlled probe anchoring (using a photomask), direct attachment without using cross-linkers (one-pot fashion), and short irradiation times (20 min). Using the described strategy, hybridization efficiencies up to 65% with full complementary strands were reached. The approach was evaluated by scoring single-base pair mismatches with discrimination ratios around 15. Moreover, the efficacy of the proposed DNA detection scheme is further demonstrated through the assay on a genomic target of bacterial Escherichia coli.
Assuntos
Alcenos/química , DNA/análise , Análise de Sequência com Séries de Oligonucleotídeos , Sondas de Oligonucleotídeos/química , Compostos de Sulfidrila/química , Química Click , Escherichia coli/isolamento & purificação , Fluorescência , Compostos de Organossilício/química , Tamanho da Partícula , Propriedades de Superfície , Raios UltravioletaRESUMO
The impact of the presence of nonnatural bases on the properties of oligodeoxynucleotides has been studied. First, oligodeoxynucleotides carrying 2'-deoxyzebularine were prepared, and the stability of duplexes carrying this analogue was determined by DNA melting experiments. Melting temperatures and thermodynamic data indicated the preference of 2'-deoxyzebularine for 2'-deoxyguanosine, which behaves as a 2'-deoxycytidine analogue, forming a less stable base pair due to the absence of the amino group at position 4. Moreover, the duplex-hairpin equilibrium of a self-complementary oligodeoxynucleotide carrying several natural and nonnatural bases including 2'-deoxyzebularine as a central mispair, was studied. Depending on the base present in the middle of the sequence, it is possible to affect the stability of the bimolecular duplex modulating the duplex-hairpin equilibrium. Magnesium ions were shown to stabilize preferentially the bimolecular duplex form. The results indicate the importance of the modifications and the role of cations in shifting the structural equilibrium.
Assuntos
Citidina/análogos & derivados , DNA/química , Oligodesoxirribonucleotídeos/química , Pareamento de Bases , Sequência de Bases , Citidina/síntese química , Citidina/química , DNA/genética , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Temperatura , TermodinâmicaRESUMO
The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.
Assuntos
Oligonucleotídeos/síntese química , Polietilenoglicóis/química , Catálise , Cobre/química , Ciclização , DNA/química , DNA Topoisomerases Tipo I/química , Ligantes , Estrutura Molecular , Oligonucleotídeos/química , Peptídeos/síntese química , Peptídeos/química , Soluções/química , Água/químicaRESUMO
The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.