Biology subjects

Weiss, Z.

Publications and source records attributed to Weiss, Z..

7 recordsLinked to original sources

bioRxiv2025

Population dynamics of ribozymes during in vitro selection

Ribozymes are central to models of RNA-based primordial life. Understanding how ribozymes emerge from RNA populations under changing selection pressures can help delineate the biochemical constraints and capabilities of early RNA-based biology. In vitro selection has been instrumental in isolating ribozymes with diverse functions from combinatorial populations; however, their population dynamics during selection remain poorly characterized. By analyzing high-throughput sequencing data produced from all rounds of an in vitro selection experiment, consisting of over 5 million unique sequences, we tracked how sequences rose and fell in abundance during selection. We found that the most abundant sequences emerged early and maintained their dominance, suggesting that only a few cycles of selection-amplification may be sufficient to isolate well-adapted ribozymes. Characterization of the catalytic activities of the dominant sequences in each selection round indicated that abundance did not always predict activity. This observation was reinforced through nucleotide conservation analysis and activity assays of close sequence variants of the most dominant ribozyme. Our analyses show that sequence complementarity with the substrate emerged as a common feature as early as the first round and persisted throughout, highlighting early convergence of diverse sequence populations on a beneficial catalytic strategy. By combining bioinformatics and experimental approaches, our work presents a detailed description of ribozyme population dynamics during an in vitro selection experiment and provides a high-resolution view of how functional RNA sequences compete for survival and propagation under selection pressure.

bioinformatics↗

bioRxiv2025

A ribozyme ligase that requires a 3' terminal phosphate on its RNA substrate

Ribozymes played essential roles in catalyzing metabolic processes and facilitating genome replication in primordial RNA-based life. In vitro evolution has enabled the discovery of ribozymes that catalyze diverse chemical reactions, but it is likely that many catalytic activities of RNA remain to be uncovered. Expanding our understanding of the biochemical capabilities of RNA, especially new ribozyme functions related to RNA assembly, will help to refine models of primordial genome replication and, more broadly, the origin and evolution of early life. Here, we report the discovery of a novel ribozyme ligase that catalyzes the attack of the 2'-hydroxyl group of an RNA substrate on the 5'-triphosphate of a second RNA, but only when the substrate RNA possesses a 3'-phosphate vicinal to its nucleophilic 2'-hydroxyl group. This activity emerged unexpectedly during a directed evolution experiment designed to isolate ribozyme ligases that use 5'-triphosphorylated RNA oligonucleotides as substrates. Because the 3'-phosphate group on the RNA substrate does not directly participate in the ligation reaction, it would be extremely challenging to design a selection strategy to isolate ribozyme ligases with this unique reactivity. The ligases requirement for an RNA 3'-phosphate group on the substrate resembles enzymatic mechanisms found in protein-based RNA repair pathways. We propose that ribozyme-catalyzed ligation of 3'-phosphorylated RNA could have enabled the repair of cleaved RNA strands in primordial cells. We demonstrate that our in vitro evolved ribozymes ligate themselves specifically to 3'-phosphorylated RNA fragments present in heterogenous mixtures of cellular RNA, demonstrating their potential utility as enrichment reagents for profiling RNA cleavage products in transcriptomic studies. Our findings not only report a new catalytic reactivity in RNA but also provide insights into ribozyme evolution, primordial RNA repair, and potential applications in RNA sequencing.

biochemistry↗

bioRxiv2024

Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole- to triphosphate-activation

The acquisition of new RNA functions through evolutionary processes would have been essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do ribozymes need completely new folds to support new but related functions, or is re-optimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly reactions. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have set the stage for the later evolution of the protein enzymes that use monomeric triphosphates (NTPs) for RNA synthesis.

biochemistry↗

bioRxiv2024

Structure-guided aminoacylation and assembly of chimeric RNAs

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.

biochemistry↗

bioRxiv2022

REVERSE: A user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments

Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. No single platform exists that performs all the steps necessary to generate publishable results starting with raw sequence data. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that incorporates an integrated computational pipeline through a graphical user interface, which performs both pre-processing and sequence level analyses within minutes. FASTQ files from multiple rounds are quality filtered, dereplicated, and trimmed before being analyzed by two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and tracks the enrichment of sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice. Both pipelines generate downloadable high-resolution figures. Collectively, REVERSE is a one stop-solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.

bioinformatics↗

bioRxiv2019

Mucoidy, a general mechanism for maintaining lytic phage in populations of bacteria

We present evidence that phage resistance resulting from overproduction of exopolysaccharides, mucoidy, provides a general answer to the longstanding question of how lytic viruses are maintained in populations dominated by bacteria upon which they cannot replicate. In serial transfer culture, populations of mucoid E. coli MG1655 that are resistant to lytic phages with different receptors, and thereby requiring independent mutations for surface resistance, are capable of maintaining these phages with little effect on their total density. Based on the results of our analysis of a mathematical model, we postulate that the maintenance of phage in populations dominated by mucoid cells can be attributed primarily to high rates of transition from the resistant mucoid states to susceptible non-mucoid states. Our tests with both population dynamic and single cell experiments as well as DNA sequence analysis are consistent with this hypothesis. We discuss reasons for the generalized resistance of these mucoid E. coli, and the genetic and molecular mechanisms responsible for the high rate of transition from mucoid to sensitive states responsible for the maintenance of lytic phage in mucoid populations of E. coli.

microbiology↗

bioRxiv2018

A Universal Algorithm to Detect Rare or Novel Cell Types in High-Throughput Single-Cell Gene Expression Data

Detecting rare cell types would allow early disease detection of cancers and infections, identification of new cell types, and a deepened understanding of cell differentiation. We developed a universal algorithm to identify rare cell types from a wide variety of single-cell omics data. We validated our algorithm on single-cell qPCR data from mouse hematopoietic cells and single-cell RNA-seq data from human glioblastoma tumors cells, both with expression values from an ample number of genes. We then applied our algorithm to seq-FISH data from mouse hippocampus cells containing expression values from only 121 genes. Our algorithm detected rare cell types including a putative new hippocampal cell type.\n\nAuthor summaryRare cell type detection would advance early disease diagnosis (e.g., cancer, infection), allow identification of new cell types, and increase understanding of cell differentiation. Current computational methods can detect common cell types, but it remains a challenge to detect rare cell types within cell populations, especially with expression data from a relatively small number of genes. We created a powerful algorithm to detect rare cell types in a population of cells. We validated our algorithm on data from mouse blood stem cells and human glioblastoma tumors cells. When we applied our algorithm to mouse hippocampus data containing expression values from only 121 genes, we detected a putative new brain cell type that no previous algorithm has identified. Our universal algorithm can now be applied to a wide range of data to detect the early onset of diseases and discover new cell types.

bioinformatics↗