Biology subjects

Holliger, P.

Publications and source records attributed to Holliger, P..

11 recordsLinked to original sources

bioRxiv2026

Continuous target-specific mutagenesis and rapid gene evolution by diversity-generating retroelements in Escherichia coli

Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue- specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with F-plasmid-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target- specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.

synthetic biology↗

bioRxiv2025

Generative inverse design of RNA structure and function with gRNAde

The design of RNA molecules with bespoke three-dimensional structures and functions is a central goal in synthetic biology and biotechnology. However, progress has been limited by the challenges of designing complex tertiary interactions such as pseudoknots, as well as engineering catalytic functions--problems that have remained largely intractable for automated methods. Here we present a high-throughput generative AI pipeline for inverse design of RNA structure and function. Central to the pipeline is gRNAde, an RNA language model conditioned on 3D backbone structures and sequence constraints. We have validated the gRNAde pipeline in a community-wide, blinded RNA design competition on the Eterna platform, where it proved able to design complex pseudoknotted RNAs at success rates matching that of human experts (95%), while significantly outperforming other physics- and AI-based automated algorithms (70%). We further demonstrate gRNAdes capabilities by generatively designing functional RNA polymerase ribozymes (RPR) with nearly 20% sequence divergence from the wild type RPR, discovering highly active variants at mutational distances inaccessible to rational design or adaptive walks by directed evolution. gRNAde thus provides an experimentally validated, open-source platform for automated design of complex RNA structures and accelerated engineering of complex RNA functions, providing a step towards programmable RNA catalysts and nanostructures. Open-source code: github.com/chaitjo/geometric-rna-design

synthetic biology↗

bioRxiv2024

A polymerase ribozyme that can synthesize both itself and its complementary strand

The emergence of a chemical system capable of self-replication and evolution is a critical event in the origin of life. RNA polymerase ribozymes could constitute such a system, but their large size and structural complexity hinder their self-replication and make their spontaneous emergence improbable. Here we describe QT45: a 45-nucleotide ribozyme, discovered from a random sequence pool, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate substrates. QT45 can synthesize both its complementary strand from a mix of all 64 trinucleotides and a copy of itself using 13 defined trinucleotides and one hexamer as substrates. The discovery of this complex activity in a small ribozyme suggests that polymerase ribozymes may be more abundant in RNA sequence space than anticipated, thereby facilitating the emergence of self-replication.

biochemistry↗

bioRxiv2024

Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences

DNA polymerases are complex molecular machines able to replicate genetic material using a template-driven process. While the copying function of these enzymes is well established, their ability to perform untemplated DNA synthesis is less well characterized. Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence of template. We use long-read nanopore sequencing and real-time PCR to observe the synthesis of pools of DNA products derived from a diverse set of natural and engineered DNA polymerases across varying temperatures and buffer compositions. We detail the features of the DNA fragments generated, enrichment of select sequence motifs, and demonstrate that the sequence composition of the synthesized DNA may be altered by modifying environmental conditions. This work provides an extensive data set to better discern the process of untemplated DNA polymerase activity and may support its potential repurposing as a technology for the guided synthesis of DNA sequences on the kilobase-scale and beyond.

synthetic biology↗

bioRxiv2024

Roles of dimeric intermediates in RNA-catalyzed rolling circle synthesis

The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling circle synthesis (RCS) on small circular RNA (scRNA) templates has been demonstrated. However, the structural and dynamic properties of scRNA replication and its products and intermediates have never been explored. Here we have used cryogenic electron microscopy (cryo-EM) to characterize products and intermediates relevant for RCS replication and find that these form an unexpectedly diverse group of RNA nanostructures. The main structural motif observed is a fully hybridized dimeric complex composed of two scRNAs and their complement strands resolved to 5.3 [A]. Cryo-EM also reveals higher order dimer filaments and dimer assembly intermediates suggesting a mechanism for assembly of the observed complexes. We show that the dimer complexes are stable and inhibit RNA-catalyzed RCS, but can be reactivated by addition of more scRNA templates. We propose that dimer formation may be a general property of RCS replication and speculate that the observed dimers might have benefited a primordial RNA genetic system by providing a stable "storage" form of RNA replication products and by coordinating RNA replication on both scRNA template strands.

biochemistry↗

bioRxiv2023

Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

RNA replication is considered a crucial stage in the origins of life. However, both enzymatic and non-enzymatic RNA replication cycles are impeded by the "strand separation problem" (SSP), a form of product inhibition arising from the extraordinary stability of RNA duplexes and their rapid kinetics of reannealing. Here we show that RNA trinucleotide triphosphates (triplets) can overcome the SSP by binding to and kinetically trapping dissociated RNA strands in a single-stranded form, while simultaneously serving as substrates for RNA replication by a triplet polymerase ribozyme (TPR). This enabled exponential replication of both (+) and (-) strands of double-stranded RNAs by the TPR when driven by coupled pH and freeze-thaw cycles. We demonstrate replication of a fragment of the ribozyme itself, and open-ended amplification of random RNA sequence pools over >70 cycles, with emergence of partial, distributive TPR self-replication and triplet codon drift towards a primordial genetic code. One-sentence summaryRNA trinucleotide substrates together with simple physicochemical cycles enable RNA-catalysed replication of double-stranded RNA and partial, distributive self-replication of an RNA polymerase ribozyme.

molecular biology↗

bioRxiv2022

Cryo-EM structure and functional landscape of an RNA polymerase ribozyme

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) including a variant that uses trinucleotide triphosphates (triplets) as substrates have been created by in vitro evolution and are the closest functional analogues of the replicase but the structural basis for their function is poorly understood. Here, we leverage single-particle cryo-EM and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The TPR cryo-EM structure at 5-[A] resolution reveals an RNA heterodimer comprising a catalytic and an inactive accessory subunit, where the complex resembles a left hand with thumb and fingers at a 70{degrees} angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme whereby heterodimer formation and KL interactions preorganize the TPR for optimal template binding and templated RNA synthesis activity. These results provide a foundation for a better understanding RNAs potential for self-replication.

biochemistry↗

bioRxiv2021

Rolling Circle RNA Synthesis Catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of lifes first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5 and 3 ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

biochemistry↗

bioRxiv2021

Emergence of function from single RNA sequences by Darwinian evolution

The spontaneous emergence of function from pools of random sequence RNA is widely considered an important transition in the origin of life. However, the plausibility of this hypothetical process and the number of productive evolutionary trajectories in sequence space are unknown. Here we demonstrate that function can arise starting from a single RNA sequence by an iterative process of mutation and selection. Specifically, we describe the discovery of both specific ATP or GTP aptamers - with micromolar affinity for their nucleotide ligand - starting each from a single, homopolymeric poly-A sequence flanked by conserved primer binding sites. Our results indicate that the ab initio presence of large, diverse random sequence pools is not a prerequisite for the emergence of functional RNAs and that the process of Darwinian evolution has the capacity to generate function even from single, largely unstructured RNA sequences with minimal molecular and informational complexity.

synthetic biology↗

bioRxiv2021

Hydrophobic-cationic peptides enhance RNA polymerase ribozyme activity by accretion

Accretion and the resulting increase in local concentration to enhance target stability and function is a widespread mechanism in biology (for example in the liquid-liquid demixing phases and coacervates). It is widely believed that such macromolecular aggregates (formed through ionic and hydrophobic interactions) may have played a role in the origin of life. Here, we report on the behaviour of a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) that forms insoluble aggregates, accrete RNA on their surfaces in a size-dependent manner, and thus enhance the activities of various ribozymes. At low Mg2+ concentrations ([Mg2+]: 25 mM MgCl2), the activity of a small ribozyme (hammerhead ribozyme) was enhanced by P43, while larger ribozymes (RNA polymerase ribozyme (RPR), RNase P, F1* ligase) were inhibited. In contrast, at high [Mg2+] ([≥]200 mM), the RPR activity was enhanced. Another hydrophobic-cationic peptide with a simpler sequence (K2V6: KKVVVVVV) also exhibited similar regulatory effects on the RPR activity. Furthermore, inactive RPR captured on P43 aggregates at low [Mg2+] could be reactivated in a high [Mg2+] buffer. Therefore, in marked contrast to previously studied purely cationic peptides (like K10) that enhance RPR only at low ionic strength, hydrophobic-cationic peptides can reversibly concentrate RNA and enhance the RPR activity even at high ionic strength conditions such as in eutectic ice phases. Such peptides could have aided the emergence of longer and functional RNAs in a fluctuating environment (e.g., dry-wet / freeze-thaw cycles) on the prebiotic earth.

biochemistry↗

bioRxiv2018

Innate potential of random genetic oligomer pools for recombination

The spontaneous emergence of function from prebiotic pools of informational polymers is a central conjecture of current origin of life scenarios. However, the innate functional capacity of random genetic polymer pools is unknown. Here, we have examined the ab initio activity of random and semi-random eicosamer pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids) with respect to a simple functional test: the capacity for intermolecular ligation and recombination. While DNA, ANA and HNA pools proved inert, naive RNA and AtNA pools displayed diverse modes of intermolecular recombination in eutectic ice phases. Recombination appears linked to the vicinal ring cis-diol shared by RNA and AtNA. Thus, the chemical configuration that renders both susceptible to hydrolysis also enables substantial spontaneous intrapool recombination in the absence of activation chemistry with a concomitant increase in the compositional and structural complexity of recombined pools.

biochemistry↗