Abstract
Protein-mediated self-assembly is arguably one of the most promising routes for building complex molecular nanostructures. Here, we report a molecular self-assembly technique that allows programmable, site-specific patterning of double-stranded DNA scaffolds, at a single-base resolution, by 3-nm-long RecA-based nucleoprotein filaments. RecA proteins bind to single-stranded DNA to form nucleoprotein filaments. These can self-assemble onto a double-stranded DNA scaffold at a region homologous to the nucleoprotein’s single-stranded DNA sequence. We demonstrate that nucleoprotein filaments can be formed from single-stranded DNA molecules ranging in length from 60 nucleotides down to just 6 nucleotides, and these can be assembled site-specifically onto a model DNA scaffold both at the end of the scaffold and away from the end. In both cases, successful site-specific self-assembly is demonstrated even for the smallest nucleoprotein filaments, which are just 3 nm long, comprise only two monomers of RecA, and cover less than one helical turn of the double-stranded DNA scaffold. Finally, we demonstrate that the RecA-mediated assembly process is highly site-specific and that the filaments indeed bind only to the homologous region of the DNA scaffold, leaving the neighboring scaffold exposed.
Highlights
Protein-mediated self-assembly is arguably one of the most promising routes for building complex molecular nanostructures
We show that nucleoprotein filaments formed from only 6-nucleotidelong ssDNA molecules (i.e., 3 nm in length) can assemble onto homologous regions on dsDNA scaffolds, covering significantly less than one helical turn of the DNA
Successful assembly can be seen even for nanoscale nucleoprotein filaments. These results demonstrate that even 3-nm-long nucleoprotein filaments can be self-assembled successfully onto DNA scaffolds, which is a significant improvement over previously established limits, where successful assembly of recombinase A (RecA) nucleoprotein filaments was observed on circular dsDNA only with the filaments longer than 15 bases and on linear dsDNA scaffolds with at least 18-base-long nucleoprotein filaments
Summary
Protein-mediated self-assembly is arguably one of the most promising routes for building complex molecular nanostructures. Molecular self-assembly is inherent to a number of biological molecules and has been exploited widely over the past few years Molecules such as DNA have been explored for the fabrication of two- and three-dimensional self-assembled DNA nanostructures.1À4 Such structures have been used as scaffolds to template and assemble complex molecular-scale objects, for example, to construct arrays of metallic nanoparticles to form molecular-scale nanowires.[5,6]. These techniques generally do not offer flexible site-specific assembly and do not allow the scaffold to be patterned programmably with high spatial accuracy. DNA nucleoprotein filaments have been explored.7À10 RecA is the major protein responsible for the homologous recombination in Escherichia coli,[11] a proteinmediated process by which a single-stranded (ss)DNA oligonucleotide is paired through self-assembly[12] and eventually exchanged with the homologous region of one of the strands of a double-stranded (ds)DNA molecule, i.e., the region with the same or a very similar base sequence on the dsDNA.[13,14] RecA-based DNA nucleoprotein filaments designed to self-assemble at sequence-specific positions on dsDNA scaffold molecules are created by polymerizing RecA monomers around the appropriate homologous ssDNA, with one RecA monomer per three nucleotides.[15]
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