RESUMO
Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme-linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation-selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)-modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.
Assuntos
Anticorpos/imunologia , Sítios de Ligação de Anticorpos , DNA Viral/química , DNA Viral/imunologia , Conformação de Ácido Nucleico , Anticorpos/química , Bacteriófago M13/genética , Imunoglobulina G/química , Imunoglobulina G/imunologia , Microscopia de Força Atômica , Microscopia Eletrônica de TransmissãoRESUMO
Designed nanostructures formed by self-assembly of multiple DNA strands suffer from low stability at elevated temperature and under other denaturing conditions. Here, we propose a method for covalent coupling of DNA strands in such structures by the formation of disulfide bonds; this allows disassembly of the structure under reducing conditions. The dynamic chemistry of disulfides and thiols was applied to crosslink DNA strands with terminal disulfide modifications. The formation of disulfide-linked DNA duplexes consisting of three strands is demonstrated, as well as a more-complex DNA double-crossover tile. All the strands in the fully disulfide-linked structures are covalently and geometrically interlocked, and it is demonstrated that the structures are stable under heating and in the presence of denaturants. Such a reversible system can be exploited in applications where higher DNA stability is needed only temporarily, such as delivery of cargoes to cells by DNA nanostructures.
Assuntos
Dissulfetos/química , Nanoestruturas/química , Oligonucleotídeos/química , DNA/síntese química , DNA/química , Eletroforese em Gel de PoliacrilamidaRESUMO
Rectangular DNA origami functionalized with thiols in each of the four corners immobilizes by self-assembly between lithographically patterned gold nanodots on a silicon oxide surface.
