Expanding RNA binding specificity and affinity of engineered PUF domains

Nucleic Acids Research
Yang-Yang ZhaoJia-Wei Wu

Abstract

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

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Citations

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

BETA
three-hybrid
PCR
gel filtration
electrophoresis
transfection

Software Mentioned

Phaser
MR
Image Quant
HKL2000
Mol
CCP4i suite
Probity
Phenix
Coot
Origin

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