RESUMO
DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX.
Assuntos
DNA/química , Microscopia de Força Atômica/métodos , Técnica de Seleção de Aptâmeros/métodos , Albumina Sérica/química , Humanos , Ligação de Hidrogênio , Ressonância de Plasmônio de SuperfícieRESUMO
Capsid-like particles consisting of a hepatitis B core (HBc) protein have been studied for their potential as carriers for drug delivery systems (DDS). The hollow HBc particle, which is formed by the self-assembly of core proteins comprising 183 aa residues, has the ability to bind to various cells non-specifically via the action of an arginine-rich domain. In this study, we developed an engineered HBc particle that specifically recognizes and targets human epidermal growth factor receptor-related 2 (HER2)-expressing breast cancer cells. To despoil the non-specific binding property of an HBc particle, we genetically deleted the C-terminal 150-183 aa part of the core protein that encodes the arginine-rich domain (ΔHBc). Then, we genetically inserted a Z(HER2) affibody molecule into the 78-81 aa position of the core protein to confer the ability of target-cell-specific recognition. The constructed Z(HER2)-displaying HBc (Z(HER2)-ΔHBc) particle specifically recognized HER2-expressing SKBR3 and MCF-7 breast cancer cells. In addition, the Z(HER2)-ΔHBc particle exhibited different binding amounts in accordance with the HER2 expression levels of cancer cells. These results show that the display of other types of affibody molecules on HBc particles would be an expandable strategy for targeting several kinds of cancer cells that would help enable a pinpoint DDS.
Assuntos
Vírus da Hepatite B/metabolismo , Engenharia de Proteínas/métodos , Receptor ErbB-2/metabolismo , Proteínas do Core Viral/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Células HeLa , Vírus da Hepatite B/genética , Humanos , Células MCF-7 , Microscopia de Força Atômica , Microscopia Confocal , Mutação , Ligação Proteica , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Proteínas do Core Viral/genéticaRESUMO
Atomic force microscopy (AFM) can dynamically detect the adhesion or affinity force between a sample surface and a cantilever. This feature is useful as a detection method using aptamers--single-strand DNA that recognizes its target with very high affinity. The present study proposes a novel DNA aptamer-based sensing system using AFM. In this study, thrombin was chosen as the target molecule, and a DNA aptamer-based AFM sensing system based on competition was developed. The affinity force between the gold chip and the cantilever decreased as the concentration of thrombin increased. Moreover, a low detection limit of 0.2 nM was achieved. Therefore, the AFM sensing system used would be appropriate for the measurement of various chemical compounds.