Periodic Reporting for period 1 - MUTAnTS (A microfluidic high throughput approach to helicase biophysics)
Reporting period: 2018-02-01 to 2020-01-31
DNA is the molecule used to store genetic information. In the double-helical structure of DNA the information-bearing nitrogenous bases face inwards. In order to access the information it is often necessary to disrupt the native DNA structure. A specific type of enzymes, helicases, use the energy of nucleotide hydrolysis to unwind the double helix for a range of biological functions, such as DNA repair and replication but also for chaperone functions. In some cases a single-enzyme is able to both unwind the double helix (helicase activity) and to copy it (polymerase activity). DNA replication and repair are not only essential biological functions, they are key reactions in many biotechnological and biomedical processes, that include DNA synthesis and sequencing. Improving our understanding of these functions is bound to have impact both at the fundamental level and in applications. Mutagenesis i.e. characterizing enzyme variants with specific amino-acid substitutions has long been used as a technique to probe sequence/function mapping. However helicases and polymerases are large macromolecules typically comprising several hundreds of amino-acids and a systematic studies of the relevance of each amino acid residue to the enzyme’s activity has so-far been impossible. In order to study large numbers of enzyme variants (mutants) in parallel we resort to water-in-oil emulsions. Each tiny droplet in our emulsions is used as a separate compartment (test-tube) to study a specific mutant. Our approach relies on microfluidic emulsification, which produces extremely monodisperse and reproducible emulsions. With this method we can study, in a single experiment, billions of mutants, something impossible by standard methods. Our scientific questions have focused on the helicase activity possessed by some polymerase families, and in particular on the biophysical mechanism that underlies it.
We have developed a novel, completely in-vitro approach that uses fL scale water-in-oil emulsions and Next Generation Sequencing to screen large libraries for strand-displacement activity. By screening mutagenic libraries based on the Phi29 polymerase, we have characterized the impact of mutations on activity along the whole gene. These results have reproduced a long list of results, obtained by mutagenesis in over 40 years of research of Phi29, providing a stringent validation of our approach. Moreover, thanks to the systematic nature of our approach, we have highlighted novel sequence features, that had escaped to previous studies, some of which can be related to strand-displacement activity. These findings have been validated screening a second mutagenic library based on a different, so-far untested, homolog. The consistency of the results obtained in the two cases suggest that the mechanism underlying strand displacement in these polymerases is conserved. In a third set of experiments we have tested all the possible combinatorial mutations at novel identified sequence motifs to unravel the biophysical details of the interaction between the polymerase and the DNA template. In parallel with these biophysical experiments we have taken steps to exploit the methodology in an applied protein-engineering setting.
The platform that we developed, readily adaptable to other enzymes, such as helicases with no replication activity, and has several technical advantages over competing approaches. The throughput of the method (109 variants/experiment) compares favorably to droplet-based approaches that either rely on cells or on droplet sorting. This allow us to screen far larger enzyme libraries, with considerable advantages both in fundamental research in applications. Moreover, our Next Generation Sequencing based readout allows us to associate each mutant with a specific, quantitative, ‘activity’, as opposed to binary (active/inactive) measurements based on droplet sorting.
Our results on the molecular basis of helicase activity are of direct technological relevance. Strand displacement activity is essential in important biotechnological application, such as whole genome amplification and DNA sequencing and the ability to design enzymes with improved strand displacement activity will have a direct impact.
Our results on the molecular basis of helicase activity are of direct technological relevance. Strand displacement activity is essential in important biotechnological application, such as whole genome amplification and DNA sequencing and the ability to design enzymes with improved strand displacement activity will have a direct impact.