Machine learning-driven protein engineering: a case study in computational drug discovery.

Research and development in drug discovery will need to find significant efficiency gains if the industry is to continue generating novel drugs. There is great expectation for machine learning (ML) to provide this boost in R&D productivity, but to harness the full potential of ML, the generation of new, high-quality datasets will be necessary. Here, the authors present a platform that combines high-throughput display and selection data generation with ML.

Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases.

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared.

XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases.

The B-family polymerases of hyperthermophilic archaea have proven an exceptional platform for engineering polymerases with extended substrate spectra, despite multiple mechanisms for detecting and avoiding incorporation of non-cognate substrates. These polymerases can efficiently synthesize and reverse-transcribe a number of xenonucleic acids (XNAs) that differ significantly from the canonical B-form of DNA. We present here a protocol for hexitol nucleic acid (HNA) synthesis by an engineered Thermococcus gorgonarius polymerase variant, including adaptation for large-scale synthesis and purification, and for other XNAs.

Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers.

Although several synthetic or xenobiotic nucleic acids (XNAs) have been shown to be viable genetic materials in vitro, major hurdles remain for their in vivo applications, particularly orthogonality. The availability of XNAs that do not interact with natural nucleic acids and are not affected by natural DNA processing enzymes, as well as specialized XNA processing enzymes that do not interact with natural nucleic acids, is essential. Here, we report 3'-2' phosphonomethyl-threosyl nucleic acid (tPhoNA) as a novel XNA genetic material and a prime candidate for in vivo XNA applications.

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis.

Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations - particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2'-5' Linkages.

Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity.

Selection of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity.

Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers.

Catalysts from synthetic genetic polymers.

The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known.

A short adaptive path from DNA to RNA polymerases.

DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain.

Synthetic genetic polymers capable of heredity and evolution.

Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures.

The Scottish Structural Proteomics Facility: targets, methods and outputs.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here.