Exploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design

Scientific Reports
Simona De FrancoMarylène Vandevenne

Abstract

Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells...Continue Reading

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Citations

Nov 5, 2019·Wiley Interdisciplinary Reviews. RNA·Carl R ShotwellJ Andrew Berglund

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Methods Mentioned

BETA
phage-display
X-ray
SELEX
Isothermal Titration Calorimetry
phage display
size exclusion chromatography
Fluorescence
gel filtration
in vitro transcription
PCR

Software Mentioned

Adobe Illustrator
Image [UNK]
WebLogo
Origin7
MicroCal
MEME
MEME ( Multiple Em for Motif Elicitation )
Octet

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