Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA.

Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2'-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control 'gate' in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously described RNA polymerase activity unlocks the synthesis of 2'-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750 nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2'OMe-RNA (2'OMezymes) for the allele-specific cleavage of oncogenic KRAS (G12D) and ß-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2'OMe-/MOE-RNA aptamers with high affinity for vascular endothelial growth factor. Our results open up these 2'-modified RNAs-used in several approved nucleic acid therapeutics-for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology.

Modified nucleic acids: replication, evolution, and next-generation therapeutics.

Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity.

The ability of reverse transcriptases (RTs) to synthesize a complementary DNA from natural RNA and a range of unnatural xeno nucleic acid (XNA) template chemistries, underpins key methods in molecular and synthetic genetics. However, RTs have proven challenging to discover and engineer, in particular for the more divergent XNA chemistries. Here we describe a general strategy for the directed evolution of RT function for any template chemistry called compartmentalized bead labelling and demonstrate it by the directed evolution of efficient RTs for 2'-O-methyl RNA and hexitol nucleic acids and the discovery of RTs for the orphan XNA chemistries D-altritol nucleic acid and 2'-methoxyethyl RNA, for which previously no RTs existed. Finally, we describe the engineering of XNA RTs with active exonucleolytic proofreading as well as the directed evolution of RNA RTs with very high complementary DNA synthesis fidelities, even in the absence of proofreading.

A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids.

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. This polyelectrolyte structure decouples information content (base sequence) from bulk properties, such as solubility, and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl phosphonate nucleic acids (phNAs) in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl and P-ethyl phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence mixed P-methyl/P-ethyl phNA repertoires. Our results establish an example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel functional molecules.

Engineering and application of polymerases for synthetic genetics.

Organic chemistry has systematically probed the chemical determinants of function in nucleic acids by variation to the nucleobase, sugar ring and backbone moieties to build synthetic genetic polymers. Concomitantly, protein engineering has advanced to allow the discovery of polymerases capable of utilizing modified nucleotide analogs. A conjunction of these two lines of investigation in nucleotide chemistry and molecular biology has given rise to a new field of synthetic genetics dedicated to the exploration of the capacity of these novel, synthetic nucleic acids for the storage and propagation of genetic information, for evolution and for crosstalk, that is, information exchange with the natural genetic system. Here we summarize recent progress in synthetic genetics, specifically in the design of novel unnatural basepairs to expand the genetic alphabet as well as progress in engineering polymerases capable of templated synthesis, reverse transcription and evolution of synthetic genetic polymers.

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering.

Nucleic acids are a distinct form of sequence-defined biopolymer. What sets them apart from other biopolymers such as polypeptides or polysaccharides is their unique capacity to encode, store, and propagate genetic information (molecular heredity). In nature, just two closely related nucleic acids, DNA and RNA, function as repositories and carriers of genetic information. They therefore are the molecular embodiment of biological information. This naturally leads to questions regarding the degree of variation from this seemingly ideal "Goldilocks" chemistry that would still be compatible with the fundamental property of molecular heredity. To address this question, chemists have created a panoply of synthetic nucleic acids comprising unnatural sugar ring congeners, backbone linkages, and nucleobases in order to establish the molecular parameters for encoding genetic information and its emergence at the origin of life. A deeper analysis of the potential of these synthetic genetic polymers for molecular heredity requires a means of replication and a determination of the fidelity of information transfer. While non-enzymatic synthesis is an increasingly powerful method, it currently remains restricted to short polymers. Here we discuss efforts toward establishing enzymatic synthesis, replication, and evolution of synthetic genetic polymers through the engineering of polymerase enzymes found in nature. To endow natural polymerases with the ability to efficiently utilize non-cognate nucleotide substrates, novel strategies for the screening and directed evolution of polymerase function have been realized. High throughput plate-based screens, phage display, and water-in-oil emulsion technology based methods have yielded a number of engineered polymerases, some of which can synthesize and reverse transcribe synthetic genetic polymers with good efficiency and fidelity. The inception of such polymerases demonstrates that, at a basic level at least, molecular heredity is not restricted to the natural nucleic acids DNA and RNA, but may be found in a large (if finite) number of synthetic genetic polymers. And it has opened up these novel sequence spaces for investigation. Although largely unexplored, first tentative forays have yielded ligands (aptamers) against a range of targets and several catalysts elaborated in a range of different chemistries. Finally, taking the lead from established DNA designs, simple polyhedron nanostructures have been described. We anticipate that further progress in this area will expand the range of synthetic genetic polymers that can be synthesized, replicated, and evolved providing access to a rich sequence, structure, and phenotypic space. "Synthetic genetics", that is, the exploration of these spaces, will illuminate the chemical parameter range for en- and decoding information, 3D folding, and catalysis and yield novel ligands, catalysts, and nanostructures and devices for applications in biotechnology and medicine.

Towards applications of synthetic genetic polymers in diagnosis and therapy.

Aptamers are a class of single-stranded nucleic acid ligands that can bind their targets with high specificity and affinities rivalling those of antibodies. First described over 20 years ago by Tuerk & Gold [1] and Ellington & Szostak [2] (who coined the name), their promise as both diagnostic and therapeutic agents remains to be realised. Key problems include the generally low biostability of the standard DNA/RNA or mixed RNA/2'F-DNA backbones under physiological conditions, limited chemical diversity of functional groups on the natural nucleobases, and the difficulty in reliably discovering aptamer ligands to some therapeutic targets. This review will describe recent progress in developing aptamer selection technology as well as expanding aptamer chemistry and informational complexity to improve aptamer discovery and properties.

Compartmentalized Self-Tagging for In Vitro-Directed Evolution of XNA Polymerases.

Template-dependent synthesis of xenobiotic nucleic acids (XNAs) is an essential step for the development of functional XNA molecules, as it enables Darwinian evolution to be carried out with novel genetic polymers. The extraordinary substrate specificity of natural DNA polymerases greatly restricts the spectrum of XNAs available, thus making it necessary to identify DNA polymerase variants capable of incorporating a wider range of substrates. This unit summarizes compartmentalized self-tagging (CST), a directed evolution strategy developed for the selection of DNA polymerase variants capable of XNA synthesis.