Real-Time Imaging of Single-Molecule Enzyme Cascade Using a DNA Origami Raft

The dynamics of enzymes are directly associated with their functions in various biological processes. Nevertheless, the ability to image motions of single enzymes in a highly parallel fashion remains a challenge. Here, we develop a DNA origami raft-based platform for in-situ real-time imaging of enzyme cascade at the single-molecule level. The motions of enzymes are rationally controlled via different tethering modes on a two-dimensional (2D) supported lipid bilayer (SLB). We construct an enzyme cascade by anchoring catalase on cholesterol-labeled double-stranded (ds) DNA and glucose oxidase on cholesterol-labeled origami rafts. DNA functionalized with cholesterol can be readily incorporated in SLB via the cholesterol–lipid interaction. By using a total internal reflection fluorescence microscope (TIRFM), we record the moving trajectory of fluorophore-labeled single enzymes on the 2D surface: the downstream catalase diffuses freely in SLB, whereas the upstream glucose oxidase is relatively immobile. By analyzing the trajectories of individual enzymes, we find that the lateral motion of enzymes increases in a substrate concentration-dependent manner and that the enhanced diffusion of enzymes can be transmitted via the cascade reaction. We expect that this platform sheds new light on studying dynamic interactions of proteins and even cellular interactions

Designed Diblock Oligonucleotide for the Synthesis of Spatially Isolated and Highly Hybridizable Functionalization of DNA–Gold Nanoparticle Nanoconjugates

Conjugates of DNA and gold nanoparticles (AuNPs) typically exploit the strong Au–S chemistry to self-assemble thiolated oligonucleotides at AuNPs. However, it remains challenging to precisely control the orientation and conformation of surface-tethered oligonucleotides and finely tune the hybridization ability. We herein report a novel strategy for spatially controlled functionalization of AuNPs with designed diblock oligonucleotides that are free of modifications. We have demonstrated that poly adenine (polyA) can serve as an effective anchoring block for preferential binding with the AuNP surface, and the appended recognition block adopts an upright conformation that favors DNA hybridization. The lateral spacing and surface density of DNA on AuNPs can also be systematically modulated by adjusting the length of the polyA block. Significantly, this diblock oligonucleotide strategy results in DNA–AuNPs nanoconjugates with high and tunable hybridization ability, which form the basis of a rapid plasmonic DNA sensor

DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis

Circulating tumor cells (CTCs) contain molecular information on the primary tumor and can be used for predictive cancer diagnostics. Capturing rare live CTCs and their quantification in whole blood remain technically challenging. Here we report an aptamer-trigger clamped hybridization chain reaction (atcHCR) method for in situ identification and subsequent cloaking/decloaking of CTCs by porous DNA hydrogels. These decloaked CTCs were then used for live cell analysis. In our design, a DNA staple strand with aptamer-toehold biblocks specifically recognizes epithelial cell adhesion molecule (EpCAM) on the CTC surface that triggers subsequent atcHCR via toehold-initiated branch migration. Porous DNA hydrogel based-cloaking of single/cluster of CTCs allows capturing of living CTCs directly with minimal cell damage. The ability to identify a low number of CTCs in whole blood by DNA hydrogel cloaking would allow high sensitivity and specificity for diagnosis in clinically relevant settings. More significantly, decloaking of CTCs using controlled and defined chemical stimuli can release living CTCs without damages for subsequent culture and live cell analysis. We expect this liquid biopsy tool to open new powerful and effective routes for cancer diagnostics and therapeutics