Discovering pathways through ribozyme fitness landscapes using information theoretic quantification of epistasis.

The identification of catalytic RNAs is typically achieved through primarily experimental means. However, only a small fraction of sequence space can be analyzed even with high-throughput techniques. Methods to extrapolate from a limited data set to predict additional ribozyme sequences, particularly in a human-interpretable fashion, could be useful both for designing new functional RNAs and for generating greater understanding about a ribozyme fitness landscape. Using information theory, we express the effects of epistasis (i.e., deviations from additivity) on a ribozyme. This representation was incorporated into a simple model of the epistatic fitness landscape, which identified potentially exploitable combinations of mutations. We used this model to theoretically predict mutants of high activity for a self-aminoacylating ribozyme, identifying potentially active triple and quadruple mutants beyond the experimental data set of single and double mutants. The predictions were validated experimentally, with nine out of nine sequences being accurately predicted to have high activity. This set of sequences included mutants that form a previously unknown evolutionary "bridge" between two ribozyme families that share a common motif. Individual steps in the method could be examined, understood, and guided by a human, combining interpretability and performance in a simple model to predict ribozyme sequences by extrapolation.

Effect of montmorillonite K10 clay on RNA structure and function.

One of the earliest living systems was likely based on RNA ("the RNA world"). Mineral surfaces have been postulated to be an important environment for the prebiotic chemistry of RNA. In addition to adsorbing RNA and thus potentially reducing the chance of parasitic takeover through limited diffusion, minerals have been shown to promote a range of processes related to the emergence of life, including RNA polymerization, peptide bond formation, and self-assembly of vesicles. In addition, self-cleaving ribozymes have been shown to retain activity when adsorbed to the clay mineral montmorillonite. However, simulation studies suggest that adsorption to minerals is likely to interfere with RNA folding and, thus, function. To further evaluate the plausibility of a mineral-adsorbed RNA world, here we studied the effect of the synthetic clay montmorillonite K10 on the malachite green RNA aptamer, including binding of the clay to malachite green and RNA, as well as on the formation of secondary structures in model RNA and DNA oligonucleotides. We evaluated the fluorescence of the aptamer complex, adsorption to the mineral, melting curves, Förster resonance energy transfer interactions, and 1H-NMR signals to study the folding and functionality of these nucleic acids. Our results indicate that while some base pairings are unperturbed, the overall folding and binding of the malachite green aptamer are substantially disrupted by montmorillonite. These findings suggest that minerals would constrain the structures, and possibly the functions, available to an adsorbed RNA world.

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation.

Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher's fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters.

Characterizing genotype-phenotype relationships of biomolecules (e.g. ribozymes) requires accurate ways to measure activity for a large set of molecules. Kinetic measurement using high-throughput sequencing (e.g. k-Seq) is an emerging assay applicable in various domains that potentially scales up measurement throughput to over 106 unique nucleic acid sequences. However, maximizing the return of such assays requires understanding the technical challenges introduced by sequence heterogeneity and DNA sequencing. We characterized the k-Seq method in terms of model identifiability, effects of sequencing error, accuracy and precision using simulated datasets and experimental data from a variant pool constructed from previously identified ribozymes. Relative abundance, kinetic coefficients, and measurement noise were found to affect the measurement of each sequence. We introduced bootstrapping to robustly quantify the uncertainty in estimating model parameters and proposed interpretable metrics to quantify model identifiability. These efforts enabled the rigorous reporting of data quality for individual sequences in k-Seq experiments. Here we present detailed protocols, define critical experimental factors, and identify general guidelines to maximize the number of sequences and their measurement accuracy from k-Seq data. Analogous practices could be applied to improve the rigor of other sequencing-based assays.

Controlled phage therapy by photothermal ablation of specific bacterial species using gold nanorods targeted by chimeric phages.

The use of bacteriophages (phages) for antibacterial therapy is under increasing consideration to treat antimicrobial-resistant infections. Phages have evolved multiple mechanisms to target their bacterial hosts, such as high-affinity, environmentally hardy receptor-binding proteins. However, traditional phage therapy suffers from multiple challenges stemming from the use of an exponentially replicating, evolving entity whose biology is not fully characterized (e.g., potential gene transduction). To address this problem, we conjugate the phages to gold nanorods, creating a reagent that can be destroyed upon use (termed "phanorods"). Chimeric phages were engineered to attach specifically to several Gram-negative organisms, including the human pathogens Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae, and the plant pathogen Xanthomonas campestris The bioconjugated phanorods could selectively target and kill specific bacterial cells using photothermal ablation. Following excitation by near-infrared light, gold nanorods release energy through nonradiative decay pathways, locally generating heat that efficiently kills targeted bacterial cells. Specificity was highlighted in the context of a P. aeruginosa biofilm, in which phanorod irradiation killed bacterial cells while causing minimal damage to epithelial cells. Local temperature and viscosity measurements revealed highly localized and selective ablation of the bacteria. Irradiation of the phanorods also destroyed the phages, preventing replication and reducing potential risks of traditional phage therapy while enabling control over dosing. The phanorod strategy integrates the highly evolved targeting strategies of phages with the photothermal properties of gold nanorods, creating a well-controlled platform for systematic killing of bacterial cells.

Modulation of α-Synuclein Aggregation In Vitro by a DNA Aptamer.

Protein aggregation is an important problem for human health and biotechnology, with consequences in areas ranging from neurodegenerative diseases to protein production yields. Methods to modulate protein aggregation are therefore essential. One suggested method to modulate protein aggregation is the use of nucleic acid aptamers, that is, single-stranded nucleic acids that have been selected to specifically bind to a target. Previous studies in some systems have demonstrated that aptamers may inhibit protein aggregation, including for α-synuclein, a protein implicated in synucleinopathies. However, the mechanisms by which aptamers might affect or modulate aggregation have not been fully determined. In this study, we investigated the effect of an aptamer that binds α-synuclein oligomer, T-SO508, on α-synuclein aggregation in vitro using thioflavin T to monitor aggregation kinetics, and we performed atomic force microscopy, transmission electron microscopy, and analytical ultracentrifugation to characterize intermediate structures. The results indicated that T-SO508, but not control DNA sequences, extends the lag phase of aggregation and stabilizes formation of a small non-fibrillar aggregate complex. Attempts to use the aptamer-induced complexes to seed fibril formation did not in fact accelerate aggregation, indicating that these structures are off-pathway for aggregation. This study highlights a potential mechanism by which aptamers may modulate the aggregation properties of proteins.

Promiscuous Ribozymes and Their Proposed Role in Prebiotic Evolution.

The ability of enzymes, including ribozymes, to catalyze side reactions is believed to be essential to the evolution of novel biochemical activities. It has been speculated that the earliest ribozymes, whose emergence marked the origin of life, were low in activity but high in promiscuity, and that these early ribozymes gave rise to specialized descendants with higher activity and specificity. Here, we review the concepts related to promiscuity and examine several cases of highly promiscuous ribozymes. We consider the evidence bearing on the question of whether de novo ribozymes would be quantitatively more promiscuous than later evolved ribozymes or protein enzymes. We suggest that while de novo ribozymes appear to be promiscuous in general, they are not obviously more promiscuous than more highly evolved or active sequences. Promiscuity is a trait whose value would depend on selective pressures, even during prebiotic evolution.

EasyDIVER: A Pipeline for Assembling and Counting High-Throughput Sequencing Data from In Vitro Evolution of Nucleic Acids or Peptides.

In vitro evolution is a well-established technique for the discovery of functional RNA and peptides. Increasingly, these experiments are analyzed by high-throughput sequencing (HTS) for both scientific and engineering objectives, but computational analysis of HTS data, particularly for peptide selections, can present a barrier to entry for experimentalists. We introduce EasyDIVER (Easy pre-processing and Dereplication of In Vitro Evolution Reads), a simple, user-friendly pipeline for processing high-throughput sequencing data from in vitro selections and directed evolution experiments. The pipeline takes as input raw, paired-end, demultiplexed Illumina read files. For each sample provided, EasyDIVER outputs a dereplicated list of unique nucleic acid and/or peptide sequences and their count reads.

High throughput sequencing of in vitro selections of mRNA-displayed peptides: data analysis and applications.

In vitro selection using mRNA display is currently a widely used method to isolate functional peptides with desired properties. The analysis of high throughput sequencing (HTS) data from in vitro evolution experiments has proven to be a powerful technique but only recently has it been applied to mRNA display selections. In this Perspective, we introduce aspects of mRNA display and HTS that may be of interest to physical chemists. We highlight the potential of HTS to analyze in vitro selections of peptides and review recent advances in the application of HTS analysis to mRNA display experiments. We discuss some possible issues involved with HTS analysis and summarize some strategies to alleviate them. Finally, the potential for future impact of advancing HTS analysis on mRNA display experiments is discussed.

Mapping a Systematic Ribozyme Fitness Landscape Reveals a Frustrated Evolutionary Network for Self-Aminoacylating RNA.

Molecular evolution can be conceptualized as a walk over a "fitness landscape", or the function of fitness (e.g., catalytic activity) over the space of all possible sequences. Understanding evolution requires knowing the structure of the fitness landscape and identifying the viable evolutionary pathways through the landscape. However, the fitness landscape for any catalytic biomolecule is largely unknown. The evolution of catalytic RNA is of special interest because RNA is believed to have been foundational to early life. In particular, an essential activity leading to the genetic code would be the reaction of ribozymes with activated amino acids, such as 5(4 H)-oxazolones, to form aminoacyl-RNA. Here we combine in vitro selection with a massively parallel kinetic assay to map a fitness landscape for self-aminoacylating RNA, with nearly complete coverage of sequence space in a central 21-nucleotide region. The method (SCAPE: sequencing to measure catalytic activity paired with in vitro evolution) shows that the landscape contains three major ribozyme families (landscape peaks). An analysis of evolutionary pathways shows that, while local optimization within a ribozyme family would be possible, optimization of activity over the entire landscape would be frustrated by large valleys of low activity. The sequence motifs associated with each peak represent different solutions to the problem of catalysis, so the inability to traverse the landscape globally corresponds to an inability to restructure the ribozyme without losing activity. The frustrated nature of the evolutionary network suggests that chance emergence of a ribozyme motif would be more important than optimization by natural selection.

Effect of UV Radiation on Fluorescent RNA Aptamers' Functional and Templating Ability.

Damage from ultraviolet (UV) radiation was likely to be an important selection pressure during the origin of life. RNA is believed to have been central to the origin of life and might form the basis for simple synthetic cells. Although photodamage of DNA has been extensively studied, photodamage is highly dependent on local molecular context, and damage to functional RNAs has been relatively under-studied. We irradiated two fluorescent RNA aptamers and monitored the loss of activity, folding, and the kinetics of lesion accumulation. The loss of activity differed depending on the aptamer, with the Spinach2 aptamer retaining substantial activity after long exposure times. The binding pocket was particularly susceptible to damage, and melting of the duplex regions increased susceptibility; this is consistent with the view that duplex formation is protective. At the same time, susceptibility varied greatly depending on context, thus emphasizing the importance of studying many different RNAs to understand UV hardiness.

Improved single-swab sample preparation for recovering bacterial and phage DNA from human skin and wound microbiomes.

BACKGROUND: Characterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. While sample preparation from other microbiomes (e.g., gut) has been the subject of extensive optimization, procedures for skin and wound microbiomes have received relatively little attention. Here we describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic. RESULTS: We find a substantial improvement when processing wound samples in particular; while only one-quarter of wound samples processed by a traditional method yielded sufficient DNA for downstream analysis, all samples processed using the improved method yielded sufficient DNA. Moreover, for both skin and wound samples, community analysis and viral reads obtained through deep sequencing of clinical swab samples showed significant improvement with the use of the improved method. CONCLUSION: Use of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples.

Phage therapy administered noninvasively could be effective in thin tubes subject to episodic flow despite washout: a simulation study.

Bacteriophages (phages) have been proposed as candidates for the treatment of bacterial infections in light of emerging antibiotic-resistant microorganisms. Bacterial growth within thin tubes is a particular concern, such as in urinary tract infections and colonization of catheters. However, it is not clear whether phage administration to the urinary tract or in catheters could be effective in the context of flow to the outside (i.e. voiding or saline flush). Here, we adapt a previous model of phage infection to a thin tube geometry mimicking the spatial organization of the urinary tract, including bacterial motility and episodic flow during which phages are washed out of the system. We show that density-dependent dynamics permit propagation of the phage infection and that washout has little effect on the timing of bacterial clearance. In addition, instillation of phage at the bottom ~0.1 mm of the tract is effective in our computational model, suggesting that therapeutic phage introduced non-invasively could be efficacious in such situations.

Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences.

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

Spontaneous network formation among cooperative RNA replicators.

The origins of life on Earth required the establishment of self-replicating chemical systems capable of maintaining and evolving biological information. In an RNA world, single self-replicating RNAs would have faced the extreme challenge of possessing a mutation rate low enough both to sustain their own information and to compete successfully against molecular parasites with limited evolvability. Thus theoretical analyses suggest that networks of interacting molecules were more likely to develop and sustain life-like behaviour. Here we show that mixtures of RNA fragments that self-assemble into self-replicating ribozymes spontaneously form cooperative catalytic cycles and networks. We find that a specific three-membered network has highly cooperative growth dynamics. When such cooperative networks are competed directly against selfish autocatalytic cycles, the former grow faster, indicating an intrinsic ability of RNA populations to evolve greater complexity through cooperation. We can observe the evolvability of networks through in vitro selection. Our experiments highlight the advantages of cooperative behaviour even at the molecular stages of nascent life.

Computational analysis of fitness landscapes and evolutionary networks from in vitro evolution experiments.

In vitro selection experiments in biochemistry allow for the discovery of novel molecules capable of specific desired biochemical functions. However, this is not the only benefit we can obtain from such selection experiments. Since selection from a random library yields an unprecedented, and sometimes comprehensive, view of how a particular biochemical function is distributed across sequence space, selection experiments also provide data for creating and analyzing molecular fitness landscapes, which directly map function (phenotypes) to sequence information (genotypes). Given the importance of understanding the relationship between sequence and functional activity, reliable methods to build and analyze fitness landscapes are needed. Here, we present some statistical methods to extract this information from pools of RNA molecules. We also provide new computational tools to construct and study molecular fitness landscapes.

Comprehensive experimental fitness landscape and evolutionary network for small RNA.

The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world.

Genetic drift suppresses bacterial conjugation in spatially structured populations.

Conjugation is the primary mechanism of horizontal gene transfer that spreads antibiotic resistance among bacteria. Although conjugation normally occurs in surface-associated growth (e.g., biofilms), it has been traditionally studied in well-mixed liquid cultures lacking spatial structure, which is known to affect many evolutionary and ecological processes. Here we visualize spatial patterns of gene transfer mediated by F plasmid conjugation in a colony of Escherichia coli growing on solid agar, and we develop a quantitative understanding by spatial extension of traditional mass-action models. We found that spatial structure suppresses conjugation in surface-associated growth because strong genetic drift leads to spatial isolation of donor and recipient cells, restricting conjugation to rare boundaries between donor and recipient strains. These results suggest that ecological strategies, such as enforcement of spatial structure and enhancement of genetic drift, could complement molecular strategies in slowing the spread of antibiotic resistance genes.

Selection for replicases in protocells.

We consider a world of nucleotide sequences and protocells. The sequences have the property of spontaneous self-replication. Some sequences - so-called replicases - have enzymatic activity in the sense of enhancing the replication rate of all (or almost all) sequences. In a well-mixed medium, natural selection would not favor such replicases because their presence equally benefits sequences with or without replicase activity. Here we show that protocells can select for replicases. We assume that sequences replicate within protocells and that protocells undergo spontaneous division. This leads to particular population structures which can augment the abundance of replicases. We explore various assumptions regarding replicase activity and protocell division. We calculate the error threshold that is compatible with selecting for replicases.