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).

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)n Probe for Highly Sensitive DNA Detection

“…[11] Since enzymatic labeling of DNA with target proteins has clear advantages with respect to site-specificity and protein-friendly reaction conditions, several enzymes that post-translationally modify proteins have been exploited. [12][13][14] However, the majority of previous research, including our efforts, [12] have focused on the preparation of DNA-protein conjugates with a 1:1 stoichiometry. The present study has focused on a DNA-(protein) n conjugate with 1:n stoichiometry.…”

A Facile Method for Reversibly Linking a Recombinant Protein to DNA

“…[8] Several methods for making covalent DNA-protein linkages have been developed, including bifunctional crosslinkers, [8,9] disulfide bonds [10] and click chemistry. [3] A methyltransferase can be used to form an abortive covalent complex with a modified base. [11] Cysteinemodified DNA oligomers can be coupled to recombinant intein-fusion proteins by expressed protein ligation.…”

“…Incorporation of proteins into these nanostructures is a promising way to increase their functionality: DNA structures have been used to arrange protein molecules in one, [1] two [2] or three dimensions [3] and to encapsulate them. [4] Other applications of DNA-protein conjugation include the formation of functional enzyme complexes, [5] fluorescence resonance energy transfer (FRET) systems, [6] sensitive detection by immuno-PCR [7] and the construction of protein or peptide arrays by hybridization to surface-bound DNA probes.…”

Postsynthetic DNA Modification through the Copper‐Catalyzed Azide–Alkyne Cycloaddition Reaction

“…To this tag the authors specifically attached an azide-modified isoprenoid diphosphate 1 with the help of the enzyme farnesyltransferase (PFTase, Scheme 2). [22] The obtained azide-bearing protein was then treated with a single-stranded oligonucleotide (ssODN) carrying an alkyne attached to a 5'-terminal phosphate group. To test whether the DNA sequence can still be addressed despite the presence of the protein, a counter strand containing a Texas Red label was successfully hybridized to the DNA strand attached to the protein.…”