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Article ; Online: Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions.

Hayden, Eric J

2016 Volume 106, Page(s) 97–104

Abstract: RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, ... ...

Abstract	RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis.
Language	English
Publishing date	2016-08-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2016.05.014
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Article ; Online: RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes.

Roberts, Jessica M / Beck, James D / Pollock, Tanner B / Bendixsen, Devin P / Hayden, Eric J

eLife

2023 Volume 12

Abstract: Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes ... ...

Abstract	Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.
MeSH term(s)	RNA, Catalytic/metabolism ; RNA ; Base Sequence ; Nucleotides ; Mutagenesis ; Nucleic Acid Conformation
Chemical Substances	RNA, Catalytic ; RNA (63231-63-0) ; Nucleotides
Language	English
Publishing date	2023-01-19
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.80360
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Article: Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions

Hayden, Eric J

Methods. 2016 Aug. 15, v. 106

2016

Abstract	RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis.
Keywords	algorithms ; genotype ; genotype-phenotype correlation ; high-throughput nucleotide sequencing ; models ; phenotype ; prediction ; RNA
Language	English
Dates of publication	2016-0815
Size	p. 97-104.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2016.05.014
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Article ; Online: Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation.

Bendixsen, Devin P / Pollock, Tanner B / Peri, Gianluca / Hayden, Eric J

Molecular biology and evolution

2021 Volume 38, Issue 7, Page(s) 2843–2853

Abstract: Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence ... ...

Abstract	Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.
Language	English
Publishing date	2021-03-14
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ZDB-ID	998579-7
ISSN	1537-1719 ; 0737-4038
ISSN (online)	1537-1719
ISSN	0737-4038
DOI	10.1093/molbev/msab074
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Article ; Online: Double-stemmed and split structural variants of fluorescent RNA Mango aptamers.

Herrera-Gutierrez, Jeremy / Burden, Steven J / Kobernat, Sarah E / Shults, Nicholas H / Smith, Mark / Fologea, Daniel / Hayden, Eric J

RNA (New York, N.Y.)

2023 Volume 29, Issue 9, Page(s) 1355–1364

Abstract: Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence, and small size. However, the simple structure of ... ...

Abstract	Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence, and small size. However, the simple structure of these aptamers, with a single base-paired stem capped by a G-quadruplex, can limit the sequence and structural modifications needed for many use-inspired designs. Here we report new structural variants of RNA Mango that have two base-paired stems attached to the quadruplex. Fluorescence saturation analysis of one of the double-stemmed constructs showed a maximum fluorescence that is ∼75% brighter than the original single-stemmed Mango I. A small number of mutations to nucleotides in the tetraloop-like linker of the second stem were subsequently analyzed. The effect of these mutations on the affinity and fluorescence suggested that the nucleobases of the second linker do not directly interact with the fluorogenic ligand (TO1-biotin), but may instead induce higher fluorescence by indirectly altering the ligand properties in the bound state. The effects of the mutations in this second tetraloop-like linker indicate the potential of this second stem for rational design and reselection experiments. Additionally, we demonstrated that a bimolecular mango designed by splitting the double-stemmed Mango can function when two RNA molecules are cotranscribed from different DNA templates in a single in vitro transcription. This bimolecular Mango has potential application in detecting RNA-RNA interactions. Together, these constructs expand the designability of the Mango aptamers to facilitate future applications of RNA imaging.
MeSH term(s)	Mangifera/genetics ; Mangifera/chemistry ; Mangifera/metabolism ; Aptamers, Nucleotide/chemistry ; Ligands ; Fluorescent Dyes/chemistry ; RNA/chemistry
Chemical Substances	Aptamers, Nucleotide ; Ligands ; Fluorescent Dyes ; RNA (63231-63-0)
Language	English
Publishing date	2023-06-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1241540-6
ISSN	1469-9001 ; 1355-8382
ISSN (online)	1469-9001
ISSN	1355-8382
DOI	10.1261/rna.079651.123
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Article ; Online: Phased nucleotide inserts for sequencing low-diversity RNA samples from in vitro selection experiments.

Bendixsen, Devin P / M Roberts, Jessica / Townshend, Brent / Hayden, Eric J

RNA (New York, N.Y.)

2020 Volume 26, Issue 8, Page(s) 1060–1068

Abstract: In vitro selection combined with high-throughput sequencing is a powerful experimental approach with broad application in the engineering and characterization of RNA molecules. Diverse pools of starting sequences used for selection are often flanked by ... ...

Abstract	In vitro selection combined with high-throughput sequencing is a powerful experimental approach with broad application in the engineering and characterization of RNA molecules. Diverse pools of starting sequences used for selection are often flanked by fixed sequences used as primer binding sites. These low diversity regions often lead to data loss from complications with Illumina image processing algorithms. A common method to alleviate this problem is the addition of fragmented bacteriophage PhiX genome, which improves sequence quality but sacrifices a portion of usable sequencing reads. An alternative approach is to insert nucleotides of variable length and composition ("phased inserts") at the beginning of each molecule when adding sequencing adaptors. This approach preserves read depth but reduces the length of each read. Here, we test the ability of phased inserts to replace PhiX in a low-diversity sample generated for a high-throughput sequencing based ribozyme activity screen. We designed a pool of 4096 RNA sequence variants of the self-cleaving twister ribozyme from
MeSH term(s)	DNA Transposable Elements/genetics ; High-Throughput Nucleotide Sequencing/methods ; Nucleotides/genetics ; RNA/genetics ; RNA, Catalytic ; Sequence Analysis, DNA/methods ; Sequence Analysis, RNA/methods
Chemical Substances	DNA Transposable Elements ; Nucleotides ; RNA, Catalytic ; RNA (63231-63-0)
Language	English
Publishing date	2020-04-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1241540-6
ISSN	1469-9001 ; 1355-8382
ISSN (online)	1469-9001
ISSN	1355-8382
DOI	10.1261/rna.072413.119
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Article: Haematophagous ectoparasites lower survival of and have detrimental physiological effects on golden eagle nestlings.

Dudek, Benjamin M / Henderson, Michael T / Hudon, Stephanie F / Hayden, Eric J / Heath, Julie A

Conservation physiology

2021 Volume 9, Issue 1, Page(s) coab060

Abstract: Haematophagous ectoparasites can directly affect the health of young animals by depleting blood volume and reducing energetic resources available for growth and development. Less is known about the effects of ectoparasitism on stress physiology (i.e. ... ...

Abstract	Haematophagous ectoparasites can directly affect the health of young animals by depleting blood volume and reducing energetic resources available for growth and development. Less is known about the effects of ectoparasitism on stress physiology (i.e. glucocorticoid hormones) or animal behaviour. Mexican chicken bugs (
Language	English
Publishing date	2021-08-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2721508-8
ISSN	2051-1434
ISSN	2051-1434
DOI	10.1093/conphys/coab060
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Article ; Online: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors.

Townshend, Brent / Xiang, Joy S / Manzanarez, Gabriel / Hayden, Eric J / Smolke, Christina D

Nature communications

2021 Volume 12, Issue 1, Page(s) 1437

Abstract: Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors ... ...

Abstract	Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.
MeSH term(s)	Aptamers, Nucleotide/chemistry ; Biosensing Techniques/methods ; Gene Expression/genetics ; Green Fluorescent Proteins/metabolism ; Metabolic Engineering/methods ; RNA, Catalytic/chemistry ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Synthetic Biology/methods
Chemical Substances	Aptamers, Nucleotide ; RNA, Catalytic ; Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2021-03-04
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-21716-0
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Article ; Online: Dynamic RNA Fitness Landscapes of a Group I Ribozyme during Changes to the Experimental Environment.

Peri, Gianluca / Gibard, Clémentine / Shults, Nicholas H / Crossin, Kent / Hayden, Eric J

Molecular biology and evolution

2021 Volume 39, Issue 3

Abstract: Fitness landscapes of protein and RNA molecules can be studied experimentally using high-throughput techniques to measure the functional effects of numerous combinations of mutations. The rugged topography of these molecular fitness landscapes is ... ...

Abstract	Fitness landscapes of protein and RNA molecules can be studied experimentally using high-throughput techniques to measure the functional effects of numerous combinations of mutations. The rugged topography of these molecular fitness landscapes is important for understanding and predicting natural and experimental evolution. Mutational effects are also dependent upon environmental conditions, but the effects of environmental changes on fitness landscapes remains poorly understood. Here, we investigate the changes to the fitness landscape of a catalytic RNA molecule while changing a single environmental variable that is critical for RNA structure and function. Using high-throughput sequencing of in vitro selections, we mapped a fitness landscape of the Azoarcus group I ribozyme under eight different concentrations of magnesium ions (1-48 mM MgCl2). The data revealed the magnesium dependence of 16,384 mutational neighbors, and from this, we investigated the magnesium induced changes to the topography of the fitness landscape. The results showed that increasing magnesium concentration improved the relative fitness of sequences at higher mutational distances while also reducing the ruggedness of the mutational trajectories on the landscape. As a result, as magnesium concentration was increased, simulated populations evolved toward higher fitness faster. Curve-fitting of the magnesium dependence of individual ribozymes demonstrated that deep sequencing of in vitro reactions can be used to evaluate the structural stability of thousands of sequences in parallel. Overall, the results highlight how environmental changes that stabilize structures can also alter the ruggedness of fitness landscapes and alter evolutionary processes.
MeSH term(s)	Genetic Fitness ; Mutation ; RNA ; RNA, Catalytic/genetics ; RNA, Catalytic/metabolism
Chemical Substances	GIR1 ribozyme ; RNA, Catalytic ; RNA (63231-63-0)
Language	English
Publishing date	2021-11-18
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	998579-7
ISSN	1537-1719 ; 0737-4038
ISSN (online)	1537-1719
ISSN	0737-4038
DOI	10.1093/molbev/msab373
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Article: Predicting higher-order mutational effects in an RNA enzyme by machine learning of high-throughput experimental data.

Beck, James D / Roberts, Jessica M / Kitzhaber, Joey M / Trapp, Ashlyn / Serra, Edoardo / Spezzano, Francesca / Hayden, Eric J

Frontiers in molecular biosciences

2022 Volume 9, Page(s) 893864

Abstract: Ribozymes are RNA molecules that catalyze biochemical reactions. Self-cleaving ribozymes are a common naturally occurring class of ribozymes that catalyze site-specific cleavage of their own phosphodiester backbone. In addition to their natural functions, ...

Abstract	Ribozymes are RNA molecules that catalyze biochemical reactions. Self-cleaving ribozymes are a common naturally occurring class of ribozymes that catalyze site-specific cleavage of their own phosphodiester backbone. In addition to their natural functions, self-cleaving ribozymes have been used to engineer control of gene expression because they can be designed to alter RNA processing and stability. However, the rational design of ribozyme activity remains challenging, and many ribozyme-based systems are engineered or improved by random mutagenesis and selection (
Language	English
Publishing date	2022-08-15
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2814330-9
ISSN	2296-889X
ISSN	2296-889X
DOI	10.3389/fmolb.2022.893864
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