Covalent DNA–Streptavidin Conjugates as Building Blocks for Novel Biometallic Nanostructures

Supramolecular aggregates of DNA, RNA, streptavidin, immunoglobulin, and nanocrystalline metal clusters can be generated by self-assembly on the basis of oligonucleotide hybridization (shown schematically). Following selective immunosorption on surface-immobilized antigen, the biometallic hybrid is detectable by electron microscopy.     

Semisynthetic DNA–Protein Conjugates for Biosensing and Nanofabrication

Conjugation with artificial nucleic acids allows proteins to be modified with a synthetically accessible, robust tag. This attachment is addressable in a highly specific manner by means of molecular recognition events, such as Watson–Crick hybridization. Such DNA–protein conjugates, with their combined properties, have a broad range of applications, such as in high‐performance biomedical diagnostic assays, fundamental research on molecular recognition, and the synthesis of DNA nanostructures. This Review surveys current approaches to generate DNA–protein conjugates as well as recent advances in their applications. For example, DNA–protein conjugates have been assembled into model systems for the investigation of catalytic cascade reactions and light‐harvesting devices. Such hybrid conjugates are also used for the biofunctionalization of planar surfaces for micro‐ and nanoarrays, and for decorating inorganic nanoparticles to enable applications in sensing, materials science, and catalysis.     

Photocaged Quinone Methide Crosslinkers for Light‐Controlled Chemical Crosslinking of Protein–Protein and Protein–DNA Complexes

Small‐molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo‐crosslinkers target almost all residues non‐specifically, complicating data analysis. Herein, we report photocaged quinone methide (PQM)‐based crosslinkers that target nine nucleophilic residues through Michael addition, including Gln, Arg, and Asn, which are inaccessible to existing chemical crosslinkers. PQM crosslinkers were used in vitro, in Escherichia coli, and in mammalian cells to crosslink dimeric proteins and endogenous membrane receptors. The heterobifunctional crosslinker NHQM could crosslink proteins to DNA, for which few crosslinkers exist. The photoactivatable reactivity of these crosslinkers and their ability to target multiple amino acids will enhance the use of chemical crosslinking for studies of protein–protein and protein–DNA networks and for structural biology.     

A Photochemical Switch for Controlling Protein–Protein Interactions

Incorporation of an unnatural amino acid containing a photolabile group in the side chain allows specific interactions between two proteins to be prevented. The photocaged ras protein in which Asp 38 has been substituted by its β-nitrobenzyl ester (Nb) is unable to interact with its effector protein p120–GAP (see drawing below) although it has the same intrinsic GTPase activity. After photocleavage of the Nb group, 50% of the p120–GAP-dependent GTPase activity relative to the wild-type protein is restored.     

Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

Self‐Assembly of Differently Shaped Protein–Polymer Conjugates through Modification of the Bioconjugation Site

Self‐assembly of protein–polymer block copolymers is an attractive route for preparing biocatalytic materials. To clarify the effect of bioconjugate shape on self‐assembly without changing the chemical details of either block, four different conjugation sites are selected on the surface of the model globular protein mCherry at residues 3, 108, 131, and 222 to alter the colloidal shape of the bioconjugate. All four mCherry‐b‐poly(N‐isopropylacrylamide) bioconjugates show qualitatively similar phase diagrams, indicating that self‐assembly is robust with respect to changes in conjugation site. However, protein orientation has an effect on the location of the order–disorder transition concentration, and the stability of the disordered micellar phase is shown to be different for each conjugate. Differences in domain spacing also suggest changes in protein orientation within the lamellae.

     

Stabilizer‐Guided Inhibition of Protein–Protein Interactions

The discovery of novel protein–protein interaction (PPI) modulators represents one of the great molecular challenges of the modern era. PPIs can be modulated by either inhibitor or stabilizer compounds, which target different though proximal regions of the protein interface. In principle, protein–stabilizer complexes can guide the design of PPI inhibitors (and vice versa). In the present work, we combine X‐ray crystallographic data from both stabilizer and inhibitor co‐crystal complexes of the adapter protein 14‐3‐3 to characterize, down to the atomic scale, inhibitors of the 14‐3‐3/Tau PPI, a potential drug target to treat Alzheimer’s disease. The most potent compound notably inhibited the binding of phosphorylated full‐length Tau to 14‐3‐3 according to NMR spectroscopy studies. Our work sets a precedent for the rational design of PPI inhibitors guided by PPI stabilizer–protein complexes while potentially enabling access to new synthetically tractable stabilizers of 14‐3‐3 and other PPIs.     

Synthesis of Substituted Pyridyl‐Pyrimidines as Potential Protein–Protein Interaction Inhibitors

An efficient synthetic method for the preparation of pyridyl‐pyrimidines as potential inhibitors of protein–protein interactions is described. The key transformation is the reaction of a pyrimidine enaminone with phenyl ethyl acetate and NH⁴OAc to yield the desired pyridyl‐pyrimidine.