A Universal Method for the Preparation of Covalent Protein–DNA Conjugates for Use in Creating Protein Nanostructures

Abstract

DNA has numerous attractive features as a scaffold for nanostructure assembly. Its rigidity, predictable structure, and assembly through complementary hybridization allow DNA to form nanoscale architectures such as cubes, [1] tetrahedra, [2] octahedra, [3,4] and 2D arrays. [5][6][7][8] By introducing proteins into DNA nanostructures, the recognition elements and functionalities that are inherent in proteins can be organized into nanostructured motifs. DNA-scaffolded protein assemblies have been used in immuno-PCR detection methods (PCR = polymerase chain reaction) [9][10][11] to arrange biocatalysts in a series for sequential reactions [12,13] and to organize other nanomaterials. [14] There are currently several methodologies used to link proteins to DNA. Proteins have been assembled onto DNA scaffolds through intervening adapter molecules such as streptavidin [12,[15][16][17][18][19][20] or aptamers. [21,22] Alternately, direct covalent conjugation can be achieved by modification of cysteine or lysine residues [23][24][25][26] or intein modification. [11,27,28] Niemyer and co-workers have employed these protein-DNA conjugates to form fluorescence resonant energy transfer (FRET) systems for use in nanobiotechnology. [29,30] Herein, we demonstrate a fusion-based strategy to regioselectively and covalently label proteins at the C terminus with single-stranded DNA. These protein-oligonucleotide chimeras were then spontaneously assembled into nanoarchitectures by complementary hybridization of the DNA. The covalent attachment strategy described herein yields a short and compact linkage between the protein and DNA molecule that allows for precise control over protein spacing and orientation in the final nanostructure.To achieve selective protein labeling, we use the enzyme protein farnesyltransferase (PFTase) to label a substrate protein containing a C-terminal tetrapeptide tag with an azide-modified isoprenoid diphosphate (1, Scheme 1).

Conformational flexibility facilitates self-assembly of complex DNA nanostructures

“…It has been used to demonstrate that DNA conformational flexibility could be exploited to assemble nanostructures that are otherwise difficult to prepare. DNA nanocages have attracted significant research efforts (9-14, 16, 30) not only because they are interesting structures, but also because they could potentially serve as encapsulation agents (31,32), nanoreactors (33), and organizational scaffolds (34,35).…”

A Straightforward Preparation of Aminoglycoside–Dinucleotide and –diPNA Conjugates via Click Ligation Assisted by Microwaves

“…[30] Here, we describe a straightforward alternative approach to obtain aminoglycoside-nucleotide conjugates via CuAAC, by coupling azido-aminoglycosides with alkynyl dinucleotides in aqueous solution (Scheme 2). The reasons for this choice were that alkynyl oligonucleotides can be readily obtained by solid-phase synthesis, [31] and several methods are reported to yield azido-aminoglycosides. [27] Furthermore, due to the orthogonality of azides and alkyne functions, no additional protection was needed for the synthetic precursors and ligation could be performed in water, where the reagents and products are highly soluble.…”

“…[22,31] Alkynyl dinucleotides were finally deprotected and cleaved from the resin by treatment with aq. conc.…”

DNA Nanotechnology: From Biology and Beyond

“…To this end, DNA lattices have been used to organize proteins, such as streptavidin (Park et al 2005), thrombin , and RuvA (Malo et al 2005), and to organize nucleic acid enzymes (DNAzymes; Garibotti et al 2006). Templating can also occur by volume or area, instead of by arrangement, as DNA tetrahedra have been used to encapsulate proteins (Erben et al 2006;Duckworth et al 2007). …”