Designs for the self-assembly of open and closed macromolecular structures and a molecular switch using DNA methyltransferases to order proteins on nucleic acid scaffolds

Abstract

The methyltransferase-directed addressing of fusion proteins to DNA scaffolds offers an approach to the construction of protein/nucleic acid biostructures with potential in a variety of applications. The technology is currently only limited by the yield of high occupancy structures. However, current evidence shows that DNA scaffolds that contain three or four targeted proteins can be reliably constructed. This permits a variety of macromolecular designs, several of which are given in this paper. Designs for open and closed two-dimensional and three-dimensional assemblies and a design for a molecular switch are discussed. The closed two-dimensional assembly takes the form of a square, and could find application as a component of other systems including a macromolecular rotaxane. The closed three-dimensional system takes the form of a trigonal bipyramid and could find application as a macromolecular carcerand. The molecular switch could find application as a peptide biosensor. Guidelines for the construction and structural verification of these designs are reported.

Design and analysis of nanoscale bioassemblies

“…The linkage between the DNA and methyltransferase is covalent, so the assembly is very stable; the linking reaction takes place at 37C, where most proteins are stable and the fusion proteins can be placed at preselected sites on a DNA scaffold of almost arbitrary structure. The most significant advantage is the possibility of creating methyltransferase fusion proteins containing peptides that bind to receptors for directed targeting of the nanostructure (46). Immobilization of linking proteins on two different Y-junctions could also lead to the formation of a carcerand (46,47).…”

“…The most significant advantage is the possibility of creating methyltransferase fusion proteins containing peptides that bind to receptors for directed targeting of the nanostructure (46). Immobilization of linking proteins on two different Y-junctions could also lead to the formation of a carcerand (46,47). For this system to be used successfully, the protein fused to the methyltransferase must be expressible as a fusion, and it is also necessary to produce significant amounts of the building blocks.…”

Mobility-shift analysis with microfluidics chips

“…We conclude that the Bioanalyzer 2100 and the DNA LabChip system, although designed for accurate and rapid sizing of DNA fragments, can also be effectively applied to the rapid detection of protein-DNA mobility shifts of interest in molecular biology, as well as the complex supramolecular aggregates now emerging from bionanoscience (7,8,10,13,(19)(20)(21)(22)(23)(24)(25)(26)(27).…”

“…We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. Achieving these goals will involve the construction of several types of biosensors and the development of programmable devices (7,8) with the complexity of prebiotic metabolosomes (9) or even small viruses. In order to make progress toward these goals, enabling tools for the rapid structural analysis of these systems must be developed.…”

Nucleoprotein Assemblies for Cellular Biomarker Detection

“…We have described an implementation of a general technology for the design and construction of nucleoprotein assemblies that has a broad spectrum of potential applications in molecular science. Many of these applications have been described previously. ,,− The nanodevice displaying three symmetrically clustered copies of thioredoxin described here, models a new class of assembly that can be envisioned for peptide and protein-ligand-based applications in cell and tumor biology as well as molecular science. For example, the expected increase in avidity afforded by multiple ligands suggests that the device could be useful in sorting cells based on surface marker expression type.…”