DNA methylation (DNAm) is an essential silencing epigenetic mark in eukaryotes. In plants, its establishment is understood, but the downstream mechanisms remain unclear. Some pathways are known, including the METHYL-BINDING DOMAIN (MBD) proteins, but they do not account for all DNAm-dependent silencing. Preliminary data comparing a mutant for DNA methyltransferases to a mutant for all known downstream pathways of DNAm in A. thaliana support the existence of unidentified DNAm readers or downstream pathway components. Redundancy among epigenetic pathways makes it difficult to discover downstream factors, as many gene families contain multiple homologs, as seen with MBD proteins. We sought to develop a screening method using CRISPR-induced gene family mutations with cell culture to enable high-throughput screening for new pathways downstream of DNAm. Initial protoplast and cell culture steps, followed by FISH and FACS, were unsuccessful due to poor signal, prompting a methodological shift to generate cell culture using a new reporter system, HALOTag-TM1-mcherry. Continued work is required to establish and validate the multiplexed CRISPR screen, including determining the optimal multiplicity of infection for plasmid insertion into cell culture. This approach will dramatically accelerate traditional screens, enabling the identification of silencing factors in a fraction of the usual time. The screen could identify new readers and downstream pathways in A. thaliana that may be conserved in other eukaryotes, such as humans.

Self-aminoacylating ribozymes demonstrate strong potential as a bridge between nucleic acids and peptide-based biomolecules in the early origin and evolution of life. Encapsulation of these ribozymes within lipid vesicles to form protocellular systems may confer greater activity and selective advantages to functional sequences; thus, achieving high encapsulation yields is of great interest in the study of protocell evolution. Here, using a novel encapsulation protocol, we examine the encapsulation of self-aminoacylating ribozymes with high yield under low ionic strength conditions and demonstrate retained/enhanced ribozyme activity under encapsulated environments. Two vesicle systems (POPC and oleic acid-based) are used to encapsulate RNA, and encapsulation yield and ribozyme reactivity with activated amino acids are measured via fluorescence curve and gel shift assay, respectively. We demonstrate successful high-yield encapsulation of ribozymes capable of both metabolic activity and genetic information encoding. This work provides proof of concept of a prebiotically plausible protocellular system and theoretical means for compartmentalization on prebiotic earth.

Self-aminoacylating ribozymes demonstrate strong potential as a bridge between nucleic acids and peptide-based biomolecules in the early origin and evolution of life. Encapsulation of these ribozymes within lipid vesicles to form protocellular systems may confer greater activity and selective advantages to functional sequences; thus, achieving high encapsulation yields is of great interest in the study of protocell evolution. Here, using a novel encapsulation protocol, we examine the encapsulation of self-aminoacylating ribozymes with high yield under low ionic strength conditions and demonstrate retained/enhanced ribozyme activity under encapsulated environments. Two vesicle systems (POPC and oleic acid-based) are used to encapsulate RNA, and encapsulation yield and ribozyme reactivity with activated amino acids are measured via fluorescence curve and gel shift assay, respectively. We demonstrate successful high-yield encapsulation of ribozymes capable of both metabolic activity and genetic information encoding. This work provides proof of concept of a prebiotically plausible protocellular system and theoretical means for compartmentalization on prebiotic earth.

Cryogenic electron microscopy is a structural determination technique that enables near-native visualization of biomolecules at atomic resolution. After data acquisition, interpreting the resulting density map requires first fitting an existing experimental or computationally predicted model, and then manually correcting poorly resolved regions, which leads to long data processing times and possible human error. To address the need for an automated workflow, we developed CryoIDv2, a tool that identifies, retrieves, and aligns the best publicly available structural candidate into a user-provided map. The software programmatically retrieves structures from the AlphaFold database and the Protein Data Bank, uses fitting algorithms to align these candidates into the map, and then scores each fit to determine the best model. Our results indicate that this workflow can successfully identify the best candidate structure, although more optimization is needed to improve identification accuracy. With only a density map as input, CryoIDv2 minimizes manual model searching, retrieval, and alignment, thereby streamlining and accelerating modeling workflows.

Fluorous chemistry leverages the unique physical and chemical properties of perfluorinated compounds, particularly their marked immiscibility with both aqueous and organic phases, to design technology capable of interacting with biological systems while avoiding unwanted interference from surrounding biomolecules. These compounds are particularly valuable for electrochemical sensing because their chemical stability reduces signal drift over time, while their phase behavior minimizes interference from competing ions. This project investigates the synthesis and optimization of fluorous imidazolium salts for their use as ionic sites in fluorous membranes of ion-selective electrodes (ISEs). By building on prior work in fluorous tagging, this study aims to address limitations of ionic sites currently used, namely their limited solubility in the fluorous phase. Two fluorous imidazolium salts with different degrees of fluorous character were synthesized: one bearing two branched perfluorohexyl chains with a tosylate counterion, and one bearing a branched perfluorohexyl chain and one methyl substituent with an iodide counterion. Following synthesis and characterization, reaction conditions were systematically modified by varying base equivalents, reaction time, and solvent type in order to increase yield. These salts are anticipated to integrate effectively into fluorous-phase ISE membranes as ionic sites, improving overall sensitivity and durability of the sensor.

This abstract has been withheld from publication.