What Worked, What Did Not: University Instruction during a Pandemic

In this report, we discuss the experience of both lecturing and teaching laboratory classes during a pandemic at the University of Mississippi (UM). UM is a relatively rural university with approximately 20 000 students. The instructional approaches that we attempted would be significantly more difficult to implement at universities with larger class sizes, geographically more restricted with regard to climate, or more urban with confined space, yet we observed many failures, even at a rural, spacious campus. Here, we note the various models of instruction that—in our case—could be separated into three approaches: in-person (i.e., traditional face-to-face instruction), online only, and a hybrid model with some component of the two (1). We discuss our experiences of what went right and what went wrong with each approach. Given that similar approaches have been undertaken around the globe, we use this report to relate what we observed as both effective and noneffective for our style of university, with special emphasis on physical biochemical laboratory training of students

Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

Topological variants of single-strand DNA (ssDNA) structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC) and 1,3-diaza-2-oxophenoxazine (tC°), can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA) to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD) spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC° is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions

Effects of Polyamine Binding on the Stability of DNA i-Motif Structures

B-form DNA can adopt alternative structures under conditions such as superhelical duress. Alternative DNA structures are favored when there is asymmetric distribution of guanosine and cytosine on complimentary DNA strands. A guanosine-rich strand can form a four-stranded structure known as a quadruplex (G4). The complimentary cytosine-rich strand can utilize intercalating cytosine−cytosine base pairing to form a four-stranded structure known as the i-motif (iM). Both secondary structures are energetically uphill from double-strand DNA (dsDNA), meaning that additional factors are needed for their formation. Most iMs require slightly acidic conditions for structure stabilization. However, crowding agents such as polyethylene glycols and dextrans can shift the pKa of the iM to near-physiological pH ≈ 7. Nucleic acids have long been known to be bound and stabilized by polyamines such as putrescine, spermidine, and spermine. Polyamines have very high concentrations in cells (0.1−30 mM), and their binding to DNA is driven by electrostatic interactions. Polyamines typically bind in the minor groove of DNA. However, because of the unusual structure of iMs, it was unknown whether polyamines might also bind and stabilize iMs. The study described here was undertaken to analyze polyamine−iM interactions. The thermal stability and pH dependence of iM structures were determined in the presence of polyamines. In contrast to dsDNA, our results suggest that polyamines have considerably weaker interactions with iMs, as demonstrated by the minimal change in iM pH dependence and thermal stability. Our results suggest that polyamines are unlikely to provide a significant source of iM stabilization in vivo

Co-Localization of DNA i-Motif-Forming Sequences and 5-Hydroxymethyl-cytosines in Human Embryonic Stem Cells

G-quadruplexes (G4s) and i-motifs (iMs) are tetraplex DNA structures. Sequences capable of forming G4/iMs are abundant near the transcription start sites (TSS) of several genes. G4/iMs affect gene expression in vitro. Depending on the gene, the presence of G4/iMs can enhance or suppress expression, making it challenging to discern the underlying mechanism by which they operate. Factors affecting G4/iM structures can provide additional insight into their mechanism of regulation. One such factor is epigenetic modification. The 5-hydroxymethylated cytosines (5hmCs) are epigenetic modifications that occur abundantly in human embryonic stem cells (hESC). The 5hmCs, like G4/iMs, are known to participate in gene regulation and are also enriched near the TSS. We investigated genomic co-localization to assess the possibility that these two elements may play an interdependent role in regulating genes in hESC. Our results indicate that amongst 15,760 G4/iM-forming locations, only 15% have 5hmCs associated with them. A detailed analysis of G4/iM-forming locations enriched in 5hmC indicates that most of these locations are in genes that are associated with cell differentiation, proliferation, apoptosis and embryogenesis. The library generated from our analysis is an important resource for investigators exploring the interdependence of these DNA features in regulating expression of selected genes in hESC

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

AbstractThe four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs

Effects of Polyamine Binding on the Stability of DNA i‑Motif Structures

B-form DNA can adopt alternative structures under conditions such as superhelical duress. Alternative DNA structures are favored when there is asymmetric distribution of guanosine and cytosine on complimentary DNA strands. A guanosine-rich strand can form a four-stranded structure known as a quadruplex (G4). The complimentary cytosine-rich strand can utilize intercalating cytosine–cytosine base pairing to form a four-stranded structure known as the i-motif (iM). Both secondary structures are energetically uphill from double-strand DNA (dsDNA), meaning that additional factors are needed for their formation. Most iMs require slightly acidic conditions for structure stabilization. However, crowding agents such as polyethylene glycols and dextrans can shift the pKa of the iM to near-physiological pH ≈ 7. Nucleic acids have long been known to be bound and stabilized by polyamines such as putrescine, spermidine, and spermine. Polyamines have very high concentrations in cells (0.1–30 mM), and their binding to DNA is driven by electrostatic interactions. Polyamines typically bind in the minor groove of DNA. However, because of the unusual structure of iMs, it was unknown whether polyamines might also bind and stabilize iMs. The study described here was undertaken to analyze polyamine–iM interactions. The thermal stability and pH dependence of iM structures were determined in the presence of polyamines. In contrast to dsDNA, our results suggest that polyamines have considerably weaker interactions with iMs, as demonstrated by the minimal change in iM pH dependence and thermal stability. Our results suggest that polyamines are unlikely to provide a significant source of iM stabilization in vivo

Insights and Ideas Garnered from Marine Metabolites for Development of Dual-Function Acetylcholinesterase and Amyloid-β Aggregation Inhibitors

Due to the diversity of biological activities that can be found in aquatic ecosystems, marine metabolites have been an active area of drug discovery for the last 30 years. Marine metabolites have been found to inhibit a number of enzymes important in the treatment of human disease. Here, we focus on marine metabolites that inhibit the enzyme acetylcholinesterase, which is the cellular target for treatment of early-stage Alzheimer’s disease. Currently, development of anticholinesterase drugs with improved potency, and drugs that act as dual acetylcholinesterase and amyloid-β aggregation inhibitors, are being sought to treat Alzheimer’s disease. Seven classes of marine metabolites are reported to possess anti-cholinesterase activity. We compared these metabolites to clinically-used acetylcholinesterase inhibitors having known mechanisms of inhibition. We performed a docking simulation and compared them to published experimental data for each metabolite to determine the most likely mechanism of inhibition for each class of marine inhibitor. Our results indicate that several marine metabolites bind to regions of the acetylcholinesterase active site that are not bound by the clinically-used drugs rivastigmine, galanthamine, donepezil, or tacrine. We use the novel poses adopted for computational drug design of tighter binding anticholinesterase drugs likely to act as inhibitors of both acetylcholinesterase activity and amyloid-β aggregation inhibition