invis: Exploring high-dimensional RNA sequences from in vitro selection

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5′-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide–activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3′-5′ phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

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Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1705 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1705-1719 ◽

Cited By ~ 141

Author(s):

Thomas D. Schaal ◽

Tom Maniatis

Keyword(s):

In Vitro Selection ◽

Sr Proteins ◽

Mrna Splicing ◽

Sequence Motifs ◽

Rna Sequences ◽

Sr Protein ◽

Enhancer Sequence ◽

Splicing Enhancer

ABSTRACT Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

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Zn2+-dependent DNAzymes that cleave all combinations of ribonucleotides

Communications Biology ◽

10.1038/s42003-021-01738-6 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Rika Inomata ◽

Jing Zhao ◽

Makoto Miyagishi

Keyword(s):

In Vitro Selection ◽

Sequence Similarity ◽

Sequencing Analysis ◽

Sequence Specificity ◽

Rna Sequences ◽

Catalytic Core ◽

Highly Active ◽

Target Rna ◽

Deep Sequencing Analysis

AbstractAlthough several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. Here, we report the isolation of two zinc-dependent DNAzymes, ZincDz1 and ZincDz2, which exhibit compact catalytic core sequences with highly versatile hydrolysis activity. They were selected through in vitro selection followed by deep sequencing analysis. Despite their sequence similarity, each DNAzyme showed different Zn2+-concentration and pH-dependent reaction profiles, and cleaved the target RNA sequences at different sites. Using various substrate RNA sequences, we found that the cleavage sequence specificity of ZincDz2 and its highly active mutant ZincDz2-v2 to be 5′-rN↓rNrPu-3′. Furthermore, we demonstrated that the designed ZincDz2 could cut microRNA miR-155 at three different sites. These DNAzymes could be useful in a broad range of applications in the fields of medicine and biotechnology.

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Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025054118 ◽

2021 ◽

Vol 118 (21) ◽

pp. e2025054118

Author(s):

Yei-Chen Lai ◽

Ziwei Liu ◽

Irene A. Chen

Keyword(s):

High Throughput Sequencing ◽

In Vitro Selection ◽

Membrane Vesicles ◽

Bulk Solution ◽

Evolutionary Adaptation ◽

Rna Sequences ◽

Highly Active ◽

Selective Permeability ◽

Evolution Of Life

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.

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ASPECT, an LDA-Based Predictive Algorithm for In Vitro Selection

10.29007/vkrq ◽

2020 ◽

Author(s):

Puzhou Wang

Keyword(s):

In Vitro Selection ◽

Latent Dirichlet Allocation ◽

De Novo ◽

A Priori ◽

Sequencing Data ◽

Rna Sequences ◽

Predictive Algorithm ◽

Selection Experiments ◽

Functional Dna

In vitro selection enables the identification of functional DNA or RNA sequences (i.e., active sequences) out of entirely or partially random pools. Various computational tools have been developed for the analysis of sequencing data from selection experiments. However, most of these tools rely on structure-function relationship that is usually unknown for de novo selection experiments. This largely restricts the applications of these algorithms. In this paper, an active sequence predictor based on Latent Dirichlet allocation (LDA), ASPECT (Active Sequence PrEdiCTor), is proposed. ASPECT is independent of a priori knowledge on the structures of active sequences. Experimental results showed that ASPECT is effective.

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