DNA-binding miniproteins based on zinc fingers. Assessment of the interaction using nanopores
Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction. ; This work received support from the MINECO (SAF2013-41943- R, BFU2016-81754-ERC, CTQ2015-70698-R, SAF2014-58398-R, and SAF2016-76689-R), the Xunta de Galicia (2015-CP082, ED431C 2017/19 and Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09), the Fundación AECC (IDEAS197VAZQ), the European Union (European Regional Development Fund – ERDF), and the European Research Council (Advanced Grant No. 340055). Support of the orfeocinqa network is also acknowledged. J. R. thanks the Xunta de Galicia for a PhD fellowship; D. R.-L. is a recipient of a Ramón y Cajal Fellowship (RYC-2013-12799) ; SI
