Nucleic acid tools for detection and characterization of biological systems

Abstract

Nucleic acids, DNA and RNA, are naturally occurring biopolymers synthetized by cells to store and propagate genetic information. They can be found in eukaryotic cells, bacteria, archaea and viruses and, thanks to the development of synthetic chemistry techniques, they can be synthetized with relative ease on demand in the laboratory. DNA and RNA can form very distinct structures through Watson-Crick base pairing, where nucleobases form hydrogen bonds between the two antiparallel strands of a double helix. The programmability of base pairs can also be used to create pre-defined structures using nucleic acids as building material. One of the implementations of this, the DNA origami technique is using a long ssDNA oligo (scaffold) and hundreds of shorter oligonucleotides (staples) to bridge different regions of the scaffold together and form well defined shapes. DNA nanostructures generated this way can be used, among other things, as carriers of functional molecules to create patterns.

In paper I. we present a method to study the spatial tolerance of antibodies by using DNA origami structures to present nanoscale antigen patterns. The DNA nanopatterns were immobilized on a surface plasmon resonance set up and the binding kinetics of different antibodies were measured. We found that the IgG subclasses and isotypes studied, were able to bind bivalently to two antigens separated by distances between 3 to 17 nm, with a distinct preference showed for the 16nm distance. Different spatial tolerance profiles were observed for a monomeric IgM, and IgG antibodies with lower affinities to antigens.

In paper II. we use a DNA origami nanostructure to create different patterns of Jag1 ligand for studying the activation mechanism of the Notch signaling pathway. By treating induced pluripotent stem (iPs) cells with various Jag1 nanopatterns we found that bigger clusters of Jag1, induced more activation of the Notch receptors. This effect was further elucidated to occur because of prolonged binding of the ligand-receptor complex, leading to activation of Notch receptors in the absence of intercellular or external forces.

In paper III. we introduce a new method to synthesize DNA origami directly on magnetic beads. Our method, tested for a variety of different DNA origami structures, can achieve up to 90% yield compared to a standard folding protocol. Additionally, the same solid support can be used to functionalize the DNA origami in a one-pot-reaction and purify them from the excess of the molecules.

In paper IV. we present a protocol for detecting viral RNA in patient samples with Covid19 by circumventing the RNA extraction step, which was a bottleneck in the detection process at the beginning of the pandemic. Samples were inactivated by heat and the RT-PCR was performed directly (hid-RT-PCR). By comparing our results with the standard diagnostic method on 597 clinical samples we concluded that hid-RT-PCR is a reliable simplified and cost-efficient method that could increase diagnostic availability and subsequent decrease in spread of the virus.