RNA splicing is the process of removing introns from pre-mRNA transcripts. This process is carried out by the spliceosome, a large protein-RNA complex responsible for efficiently identifying the boundaries of introns within pre-mRNA transcripts. Besides intron excision, the spliceosome has been implicated in regulating expression of the intronless BDF2 gene in Saccharomyces cerevisiae through a pathway known as spliceosome-mediated decay (SMD). We have identified several additional intronless genes whose expression may be regulated through SMD, and which contain canonical 5' splice sites within their transcripts. We expect that inactivation of the splice sites will lead to overexpression of these targets, potentially altering cell behavior. We have developed a novel method of detecting lariats produced by SMD from these transcripts to confirm that the splice sites, in conjunction with a downstream branchpoint, are being used by the spliceosome. We can then sequence fragments of the lariats to detect unique sequences that do not exist in the linear mRNA transcript. Using this method, we have detected lariat intermediates for both BDF2 and another novel SMD target and expect to detect such intermediates for our other SMD targets. These results provide a new method of identifying SMD targets and point to the spliceosome playing a larger than expected role in gene regulation.

Self-aminoacylating ribozymes are of great interest as a potential bridge between the nucleic acid and peptide in the RNA World hypothesis, and as models for directed evolution, an emerging technique in synthetic biology. Despite their versatility in vitro, current fitness landscape mappings reveal a frustrated network, restricting evolutionary optimization. However, these mappings typically arise from experiments under fixed environmental conditions, while primitive Earth was much more environmentally dynamic, implying existing networks are incomplete. Here, we use varying temperatures to conduct ribozyme selection experiments using a doped RNA library to probe hydrolysis resistance, overall reactivity and sequence enrichment within known ribozyme families. We use experimental results to construct a more complete fitness landscape to reflect ribozyme evolutionary networks under dynamic temperature conditions. Such a model more accurately reflects the evolutionary conditions and behaviors of ribozymes on early Earth during the origins of life.

Experiments show the absorption spectrum of the hydrated electron blue-shifts in electrolyte solutions compared to in pure water. This shift has been assigned to the electron's competitive ion-pairing interactions with salt cations relative to salt anions. However, little work has been done investigating their behavior in chaotropic cation salts, which should greatly change the ion-pairing interactions given that the hydrated electron is highly chaotropic. In this work, we use mixed quantum/classical molecular dynamic simulations to analyze the behavior of two electron models in aqueous RbF and RbI solutions as a function of salt concentration. We find that the magnitude of the salt-induced spectral blue-shift directly reflects the electron’s local environment. The soft-cavity electron model predicts stronger competitive interactions with Rb+ relative to I- than a more traditional hard cavity model, leading to different predicted spectral shifts that provide a way to distinguish between the two models experimentally. At the same concentration, salts with chaotropic cations produce larger spectral blue-shifts than salts with kosmotropic cations. At high salt concentrations with chaotropic cations, the predicted blue-shift is greater when the salt anion is kosmotropic instead of chaotropic. Our goal is to inspire experimentalists to make such measurements, which will help provide a spectroscopic means to distinguish between different simulation models that predict different hydration structures for the hydrated electron.

Regulating bile acid homeostasis is a key therapeutic target for managing obesity and cholestatic liver diseases, as bile acids are essential for dietary lipid absorption. Bile acid sequestrants, commonly used to lower high bile acid levels, bind bile acids in the intestine and prevent their reabsorption. This forces the liver to convert low-density lipoprotein (LDL) cholesterol into bile acids, ultimately reducing LDL levels. However, these therapies have drawbacks, such as impaired absorption of essential medications and reduced long-term efficacy, often leading to relapse after discontinuation. In our research, we are using structure-activity relationship (SAR) analysis to design and synthesize ketoconazole derivatives as selective cytochrome P450 inhibitors. Our focus is on CYP7A1, the enzyme responsible for catalyzing the first and rate-limiting step in converting cholesterol to bile acids. The synthesis begins with an SN2 reaction between trichloroacetophenone and imidazole. From this scaffold, we are exploring synthetic strategies to modify stereoselectivity and chemoselectivity to enhance CYP7A1 specificity and potency. Through iterative SAR studies, we aim to refine these derivatives to achieve selective inhibition with minimal off-target effects. Our goal is to develop novel, targeted inhibitors of bile acid synthesis as alternative therapies for metabolic and liver disorders, overcoming the limitations of existing treatments.