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, DddA, that can act upon double-stranded DNA.

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.

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.

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.

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.

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.

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.

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.

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.