Solid Phase Synthesis of DNA Nanostructures in Heavy Liquid.

Introduction of the solid phase method to synthesize biopolymers has revolutionized the field of biological research by enabling efficient production of peptides and oligonucleotides. One of the advantages of this method is the ease of removal of excess production materials from the desired product, as it is immobilized on solid substrate. The DNA origami method utilizes the nature of nucleotide base-pairing to construct well-defined objects at the nanoscale, and has become a potent tool for manipulating matter in the fields of chemistry, physics, and biology. Here, the development of an approach to synthesize DNA nanostructures directly on magnetic beads, where the reaction is performed in heavy liquid to maintain the beads in suspension is reported. It is demonstrated that the method can achieve high folding yields of up to 90% for various DNA shapes, comparable to standard folding. At the same time, this establishes an easy, fast, and efficient way to further functionalize the DNA origami in one-pot, as well as providing a built-in purification method for easy removal of excess by-products such as non-integrated DNA strands and residual functionalization molecules.

Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force.

The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.

A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns.

The clustering of death receptors (DRs) at the membrane leads to apoptosis. With the goal of treating tumours, multivalent molecular tools that initiate this mechanism have been developed. However, DRs are also ubiquitously expressed in healthy tissue. Here we present a stimuli-responsive robotic switch nanodevice that can autonomously and selectively turn on the display of cytotoxic ligand patterns in tumour microenvironments. We demonstrate a switchable DNA origami that normally hides six ligands but displays them as a hexagonal pattern 10 nm in diameter once under higher acidity. This can effectively cluster DRs and trigger apoptosis of human breast cancer cells at pH 6.5 while remaining inert at pH 7.4. When administered to mice bearing human breast cancer xenografts, this nanodevice decreased tumour growth by up to 70%. The data demonstrate the feasibility and opportunities for developing ligand pattern switches as a path for targeted treatment.

Stochastic modeling of antibody binding predicts programmable migration on antigen patterns.

Viruses and bacteria commonly exhibit spatial repetition of surface molecules that directly interface with the host immune system. However the complex interaction of patterned surfaces with immune molecules containing multiple binding domains is poorly understood. We developed a pipeline for constructing mechanistic models of antibody interactions with patterned antigen substrates. Our framework relies on immobilized DNA origami nanostructures decorated with precisely placed antigens. The results revealed that antigen spacing is a spatial control parameter that can be tuned to influence antibody residence time and migration speed. The model predicts that gradients in antigen spacing can drive persistent, directed antibody migration in the direction of more stable spacing. These results depict antibody-antigen interactions as a computational system wherein antigen geometry constrains and potentially directs antibody movement. We propose that this form of molecular programmability could be exploited during co-evolution of pathogens and immune systems or in the design of molecular machines.

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.

Publisher Correction: Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

In the Supplementary Information file originally published with this Article, the Supplementary references 48-62 were missing; the amended file has now been uploaded.

Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

Although repetitive patterns of antigens are crucial for certain immune responses, an understanding of how antibodies bind and dynamically interact with various spatial arrangements of molecules is lacking. Hence, we introduced a new method in which molecularly precise nanoscale patterns of antigens are displayed using DNA origami and immobilized in a surface plasmon resonance set-up. Using antibodies with identical antigen-binding domains, we found that all the subclasses and isotypes studied bind bivalently to two antigens separated at distances that range from 3 to 17 nm. The binding affinities of these antibodies change with the antigen distances, with a distinct preference for antigens separated by approximately 16 nm, and considerable differences in spatial tolerance exist between IgM and IgG and between low- and high-affinity antibodies.