Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers

Abstract

This protocol describes the directed evolution of artificial endonuclease and ligase enzymes composed of synthetic genetic polymers (XNAzymes), using 'cross-chemistry selective enrichment by exponential amplification' (X-SELEX). The protocol is analogous to (deoxy)ribozyme selections, but it enables the development of fully substituted catalysts. X-SELEX is initiated by the synthesis of diverse repertoires (here 10(14) different sequences), using xeno nucleic acid (XNA) polymerases, on DNA templates primed with DNA, RNA or XNA oligonucleotides that double as substrates, allowing selection for XNA-catalyzed cleavage or ligation. XNAzymes are reverse-transcribed into cDNA using XNA-dependent DNA polymerases, and then PCR-amplified to generate templates for subsequent rounds or deep sequencing. We describe methods developed for four XNA chemistries, arabino nucleic acids (ANAs), 2'-fluoroarabino nucleic acids (FANAs), hexitol nucleic acids (HNAs) and cyclohexene nucleic acids (CeNAs), which require ∼1 week per round, and typically 10-20 rounds; in principle, these methods are scalable and applicable to a wide range of novel XNAzyme chemistries, substrates and reactions.

Selecting Fully‐Modified XNA Aptamers Using Synthetic Genetics

“…Transfer tubes/plate to thermal cycler and heat at 95°C for 30 sec, then immediately transfer to ice for 1 min. (Lim et al, 2012), or if template or primer is biotinylated, XNA can be isolated using streptavidin-coated magnetic beads (see Taylor & Holliger, 2015b); in this scenario, synthesis can be primed using RNA, which can subsequently be removed by alkaline hydrolysis].…”

“…If proceeding to further XNA synthesis, it is essential to verify the purity of this cDNA preparation (e.g., by UV absorbance), in particular to make sure that desalting and removal of ethanol are complete; carryover from previous steps could inhibit XNA polymerases. The sequence diversity of the library can be assessed by deep sequencing-we have previously described a simple method for preparing samples for sequencing using the Illumina MiSeq system (Taylor & Holliger, 2015b).…”

Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase

“…The introduction of functional diversity at the nucleic acid backbone has proven to be of importance for the development of new ligands (aptamers, siRNA; Bramsen, Grünweller, Hartmann, & Kjems, 2014;Ruckman et al, 1998), catalysts (XNAZymes; Taylor & Holliger, 2015), and nanostructures with potential applications as new materials (XNA origami; Taylor et al, 2016). Indeed, XNAs can have physicochemical properties that make them highly relevant to biomedical and biotechnological applications: Higher resistance to nucleases, increased duplex stability (to DNA, RNA, or to the XNA itself), improved pharmacokinetic properties, and reduced immunogenic potential (Bramsen et al, 2014;Pinheiro & Holliger, 2014).…”

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks

“…Some of these XNAs allowed duplex formation [67] and information storage [70,71], and were even evolved into functional molecules such as aptamers and XNAzymes [70–73]. These results suggest that DNA and RNA are not, in principle, the only genetic polymers capable of sustaining life, and so life based on XNAs may also be thinkable.…”