Evolution of a General RNA-Cleaving FANA Enzyme

Yajun Wang,John C Chaput,Arlene K Ngor,Ali Nikoomanzar

doi:10.1038/s41467-018-07611-1

Yajun Wang, John C Chaput + Show 2 more

Open Access

https://doi.org/10.1038/s41467-018-07611-1

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Journal: Nature Communications	Publication Date: Nov 29, 2018
Citations: 67	License type: open-access

Affiliation: University of California, Irvine

Abstract

The isolation of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that catalysis is not limited to natural biopolymers, but it remains unknown whether such systems can achieve robust catalysis with Michaelis-Menten kinetics. Here, we describe an efficient RNA-cleaving 2’-fluoroarabino nucleic acid enzyme (FANAzyme) that functions with a rate enhancement of >106-fold over the uncatalyzed reaction and exhibits substrate saturation kinetics typical of most natural enzymes. The FANAzyme was generated by in vitro evolution using natural polymerases that were found to recognize FANA substrates with high fidelity. The enzyme comprises a small 25 nucleotide catalytic domain flanked by substrate-binding arms that can be engineered to recognize diverse RNA targets. Substrate cleavage occurs at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Our results expand the chemical space of nucleic acid enzymes to include nuclease-resistant scaffolds with strong catalytic activity.

Highlights

The isolation of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that catalysis is not limited to natural biopolymers, but it remains unknown whether such systems can achieve robust catalysis with Michaelis-Menten kinetics
Recognizing that Fluoroarabino nucleic acid (FANA) is a close structural analog of Deoxyribose nucleic acid (DNA) (Fig. 1a)[21], we postulated that many of the critical enzyme–substrate contacts required for a DNA polymerase to recognize a DNA/DNA homoduplex would likely be maintained in a FANA/DNA heteroduplex[22]
Following the discovery of xenonucleic acids (XNAs) aptamers by in vitro selection[11,12], researchers sought to develop the first examples of XNA enzymes that could fold themselves into shapes with catalytic activity

Summary

Introduction

The isolation of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that catalysis is not limited to natural biopolymers, but it remains unknown whether such systems can achieve robust catalysis with Michaelis-Menten kinetics. 10–23 has been used to silence the expression of numerous pathological RNAs6, including targets associated with T helper type 2-driven asthma and basal-cell carcinoma[7,8], the in vitro application of this and other related enzymes are limited by the intrinsic biological stability of DNA and RNA, which are prone to nuclease digestion While this problem can be overcome with chemical modifications that are introduced post-selection[9], care must be taken not to disrupt the activity of the catalytic domain. None of the existing XNA enzymes have been shown to function with Michaelis–Menten kinetics, which suggests that their substrate binding affinity (KM) maybe unsuitable for saturation kinetics across a range of substrate concentrations This observation raises the question of whether XNAs are inherently limited in their ability to fold into complex tertiary structures capable of achieving robust catalytic activity or, alternatively, whether XNA enzymes are constrained by the enzymes used to replicate them under in vitro selection conditions.

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