A DNA-nanoassembly-based approach to map membrane protein nanoenvironments.

Most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive analysis of the compositions and spatial organizations of membrane protein nanodomains in cell populations. Here we describe the development of a non-microscopy-based method for ensemble analysis of membrane protein nanodomains. The method, termed nanoscale deciphering of membrane protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. Using NanoDeep, we characterized the nanoenvironments of Her2, a membrane receptor of critical relevance in cancer. Importantly, we were able to modulate by design the inventory of proteins analysed by NanoDeep. NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function.

A compact nanobody-DNAzyme conjugate enables antigen detection and signal amplification.

Hemin-utilizing G-quadruplex DNAzymes with peroxidase-like (POX) activity are widely used as signal reporters in biosensing technology. However, their application to protein detection has been mostly limited to sandwich-type assays involving streptavidin or nanoparticles as indirect bridging platforms between DNAzymes and antibodies. Herein, we describe the generation of a compact, covalently DNAzyme-labeled nanobody which was successfully tested in a direct enzyme-linked immunosorbent assay (ELISA). The conjugation approach was based on the self-labeling protein tag mVirD2, a truncated bacterial relaxase able to covalently bind DNA with 1:1 stoichiometry at a specific amino acid residue. The hybrid molecule combined the nanobody antigen binding affinity and specificity with the DNAzyme catalytic capability to oxidize peroxidase substrates (e.g. ABTS, H2O2). The proposed strategy is simple and cost-effective, enables development into multiplex formats and provides reagents with hitherto unmet reproducibility in terms of POX activity instrumental for both colorimetric and electrochemical reactions. As a proof-of-concept, it was demonstrated that DNAzyme-nanobody conjugates are convenient immunoreagents for rapid and specific detection of the toxic alga Alexandrium minutum.

A computational framework for DNA sequencing microscopy.

We describe a method whereby microscale spatial information such as the relative positions of biomolecules on a surface can be transferred to a sequence-based format and reconstructed into images without conventional optics. Barcoded DNA "polymerase colony" (polony) amplification techniques enable one to distinguish specific locations of a surface by their sequence. Image formation is based on pairwise fusion of uniquely tagged and spatially adjacent polonies. The network of polonies connected by shared borders forms a graph whose topology can be reconstructed from pairs of barcodes fused during a polony cross-linking phase, the sequences of which are determined by recovery from the surface and next-generation (next-gen) sequencing. We developed a mathematical and computational framework for this principle called polony adjacency reconstruction for spatial inference and topology and show that Euclidean spatial data may be stored and transmitted in the form of graph topology. Images are formed by transferring molecular information from a surface of interest, which we demonstrated in silico by reconstructing images formed from stochastic transfer of hypothetical molecular markers. The theory developed here could serve as a basis for an automated, multiplexable, and potentially superresolution imaging method based purely on molecular information.

Interplay of Three G-Quadruplex Units in the KIT Promoter.

The proto-oncogene KIT encodes for a tyrosine kinase receptor, which is a clinically validated target for treating gastrointestinal stromal tumors. The KIT promoter contains a G-rich domain within a relatively long sequence potentially able to form three adjacent G-quadruplex (G4) units, namely, K2, SP, and K1. These G4 domains have been studied mainly as single quadruplex units derived from short truncated sequences and are currently considered promising targets for anticancer drugs, alternatively to the encoded protein. Nevertheless, the information reported so far does not contemplate the interplay between those neighboring G4s in the context of the whole promoter, possibly thwarting drug-discovery efforts. Here we report the structural and functional study of the KIT promoter core sequence, in both single- and double-stranded forms, which includes all three predicted G4 units. By preventing the formation of alternatively one or two G4 units and by combining biophysical techniques and biological assays, we show for the first time that these quadruplexes cannot be analyzed independently, but they are correlated to each other. Our data suggest that, while K2 and K1 G-rich sequences retain the ability to fold into parallel G4 motifs within a long sequence, the SP G-rich domain contributes to G4 structure only together with K2. Remarkably, we have found that, in the context of a dynamic equilibrium between the three G4 units, the G4 formed by K1 has the most significant influence on the structure stability and on the biological role of the whole promoter.

Entirely enzymatic nanofabrication of DNA-protein conjugates.

While proteins are highly biochemically competent, DNA offers the ability to program, both reactions and the assembly of nanostructures, with a control that is unprecedented by any other molecule. Their joining: DNA-protein conjugates - offer the ability to combine the programmability of DNA with the competence of proteins to form novel tools enabling exquisite molecular control and the highest biological activity in one structure. However, in order for tools like these to become viable for biological applications, their production must be scalable, and an entirely enzymatic process is one way to achieve this. Here, we present a step in this direction: enzymatic production of DNA-protein conjugates using a new self-labeling tag derived from a truncated VirD2 protein of Agrobacterium tumefaciens. Using our previously reported MOSIC method for enzymatic ssDNA oligo production, we outline a pipeline for protein-DNA conjugates without the need for any synthetic chemistry in a one-pot reaction. Further, we validate HER2 staining using a completely enzymatically produced probe, enable the decoration of cell membranes and control of genetic expression. Establishing a method where protein-DNA conjugates can be made entirely using biological or enzymatic processing, opens a path to harvest these structures directly from bacteria and ultimately in-vivo assembly.

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA.

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3'-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.