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Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC50) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.

Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC

Enzymes that act on multiple substrates are common in biology but pose unique challenges as therapeutic targets. The metalloprotease insulin-degrading enzyme (IDE) modulates blood glucose levels by cleaving insulin, a hormone that promotes glucose clearance. However, IDE also degrades glucagon, a hormone that elevates glucose levels and opposes the effect of insulin. IDE inhibitors to treat diabetes, therefore, should prevent IDE-mediated insulin degradation, but not glucagon degradation, in contrast with traditional modes of enzyme inhibition. Using a high-throughput screen for non-active-site ligands, we discovered potent and highly specific small-molecule inhibitors that alter IDE's substrate selectivity. X-ray co-crystal structures, including an IDE-ligand-glucagon ternary complex, revealed substrate-dependent interactions that enable these inhibitors to potently block insulin binding while allowing glucagon cleavage, even at saturating inhibitor concentrations. These findings suggest a path for developing IDE-targeting therapeutics, and offer a blueprint for modulating other enzymes in a substrate-selective manner to unlock their therapeutic potential.

The chemical functionalities within biopolymers determine their physical properties and biological activities. The relationship between the side chains available to a biopolymer population and the potential functions of the resulting polymers, however, has proven difficult to study experimentally. Using seven sets of chemically diverse charged, polar, and nonpolar side chains, we performed cycles of artificial translation, in vitro selections for binding to either PCSK9 or IL-6 protein, and replication on libraries of random side chain-functionalized nucleic acid polymers. Polymer sequence convergence, bulk population target binding, affinity of individual polymers, and head-to-head competition among post-selection libraries collectively indicate that polymer libraries with nonpolar side chains outperformed libraries lacking these side chains. The presence of nonpolar groups, resembling functionality existing in proteins but missing from natural nucleic acids, thus may be strong determinants of binding activity. This factor may contribute to the apparent evolutionary advantage of proteins over their nucleic acid precursors for some molecular recognition tasks. A comprehensive analysis of the effect of the side chain composition of nucleic acid polymers revealed that the polarity of the side chains determines the performance of polymers during in vitro selections for protein binding.

Directed evolution can generate proteins with tailor-made activities. However, full-length genotypes, their frequencies and fitnesses are difficult to measure for evolving gene-length biomolecules using most high-throughput DNA sequencing methods, as short read lengths can lose mutation linkages in haplotypes. Here we present Evoracle, a machine learning method that accurately reconstructs full-length genotypes (R2 = 0.94) and fitness using short-read data from directed evolution experiments, with substantial improvements over related methods. We validate Evoracle on phage-assisted continuous evolution (PACE) and phage-assisted non-continuous evolution (PANCE) of adenine base editors and OrthoRep evolution of drug-resistant enzymes. Evoracle retains strong performance (R2 = 0.86) on data with complete linkage loss between neighboring nucleotides and large measurement noise, such as pooled Sanger sequencing data (~US$10 per timepoint), and broadens the accessibility of training machine learning models on gene variant fitnesses. Evoracle can also identify high-fitness variants, including low-frequency ‘rising stars’, well before they are identifiable from consensus mutations. Using short-read sequencing data, Evoracle reconstructs full-length genotypes and fitness during directed evolution experiments, and can identify low-frequency variants before they can be identified using consensus mutations.

Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase sortase A to recognize the LMVGG sequence in endogenous amyloid-β (Aβ) protein. Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aβ in human cerebrospinal fluid, enabling the detection of Aβ with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aβ42, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of sortase-mediated labeling of Aβ peptides. Laboratory evolution of the bacterial transpeptidase sortase A coupled with yeast display selection enables a change of the enzyme’s substrate preference to recognize and covalently label endogenous amyloid-β protein, impeding the protein’s ability to aggregate.

We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon-independent; the anticodon does not contact the enzyme. Using standard microbiological culture equipment, we then established a new method for laboratory evolution – a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a non-canonical amino acid.

Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.

The chemical functionalities within biopolymers determine their physical properties and biological activities. The relationship between the side-chains available to a biopolymer population and the potential functions of the resulting polymers, however, has proven difficult to study experimentally. Using seven sets of chemically diverse charged, polar, and nonpolar side-chains, we performed cycles of artificial translation, in vitro selections for binding to either PCSK9 or IL-6 protein, and replication on libraries of random side-chain-functionalized nucleic acid polymers. Polymer sequence convergence, bulk population target binding, affinity of individual polymers, and head-to-head competition among post-selection libraries collectively indicate that polymer libraries with nonpolar side-chains outperformed libraries lacking these side-chains. The presence of nonpolar groups, resembling functionality present in proteins but missing from natural nucleic acids, thus may be strong determinants of binding activity. This factor may contribute to the apparent evolutionary advantage of proteins over their nucleic acid precursors for some molecular recognition tasks.

Enzymes that act on multiple substrates are common in biology but pose unique challenges as therapeutic targets. The metalloprotease insulin-degrading enzyme (IDE) modulates blood glucose levels by cleaving insulin, a hormone that promotes glucose clearance. However, IDE also degrades glucagon, a hormone that elevates glucose levels and opposes the effect of insulin. IDE inhibitors to treat diabetes, therefore, should prevent IDE-mediated insulin degradation, but not glucagon degradation, in contrast with traditional modes of enzyme inhibition. Using a high-throughput screen for non-active-site ligands, we discovered potent and highly specific small-molecule inhibitors that alter IDE's substrate selectivity. X-ray co-crystal structures, including an IDE-ligand-glucagon ternary complex, revealed substrate-dependent interactions that enable these inhibitors to potently block insulin binding while allowing glucagon cleavage, even at saturating inhibitor concentrations. These findings suggest a path for developing IDE-targeting therapeutics, and offer a blueprint for modulating other enzymes in a substrate-selective manner to unlock their therapeutic potential.

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently discovered from a DNA-templated macrocycle library inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated gatekeeper mutant. We solved two co-crystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bi-substrate competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties, and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance.

Nat. Chem. Biol. 13, 1253–1260 (2017); published online 16 October 2017; corrected after print 1 December 2017 In the version of this article initially published, the label colors for p-NF and p-IF in the key for Figure 4c were transposed. The cyan bars should correspond to p-NF and the fuchsia barsto p-IF.

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.

Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to substantially accelerate laboratory evolution experiments. In this work, we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counterselection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates while maintaining wild type-like levels of activity.

A general MS-based screen for unusually hydrophobic cellular small molecule-RNA conjugates revealed geranylated RNA in Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella enterica var. Typhimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNA(Glu)(UUC), tRNA(Lys)(UUU) and tRNA(Gln)(UUG) at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNA(Glu)(UUC), tRNA(Lys)(UUU) and tRNA(Gln)(UUG) affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule. This technique revealed NAD-linked RNA in Escherichia coli and Streptomyces venezuelae. Subsequent characterization showed that NAD is a 5' modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs and is present at a surprisingly high abundance of approximately 3,000 copies per cell.

Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore's iron-scavenging ability in mammals.