Nucleic acid tools for detection and characterization of biological systems

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

Elastine pulmonaire humaine : etude de la degradation par les enzymes leucocytaires humains

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

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.Peer reviewe

Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) analysis. Commercial one‐step master mixes—which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well—are key reagents for SARS‐CoV‐2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M‐MLV reverse transcriptase and assemble a homemade one‐step RT‐qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS‐CoV‐2 RNA detection by RT‐qPCR: heat‐inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT‐qPCR using the homemade master mix, how to prepare in vitro−transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT‐PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one‐step RT‐qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT‐PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One‐step RT‐qPCR of RNA samples using a real‐time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One‐step RT‐PCR using endpoint fluorescence detectio