Double and triple mutant thermodynamic cycles provide a means to dissect the cooperativity of RNA and DNA folding at both the secondary and tertiary structural levels through use of the thermodynamic ...box or cube. In this article, we describe three steps for applying thermodynamic cycles to nucleic acid folding, with considerations of both conceptual and experimental features. The first step is design of an appropriate system and development of hypotheses regarding which residues might interact. Next is implementing this design in terms of a tractable experimental strategy, with an emphasis on UV melting. The final step, and the one we emphasize the most, is interpreting mutant cycles in terms of coupling between specific residues in the RNA or DNA. Coupling free energy in the absence and presence of changes elsewhere in the molecule is discussed in terms of specific folding models, including stepwise folding and concerted changes. Last, we provide a practical section on the use of commercially available software (KaleidaGraph) to fit melting data, along with a consideration of error propagation. Along the way, specific examples are chosen from the literature to illustrate the methods. This article is intended to be accessible to the biochemist or biologist without extensive thermodynamics background.
Secondary structure plays critical roles in nucleic acid function. Mismatches in DNA can lead to mutation and disease, and some mismatches involve a protonated base. Among protonated mismatches, A+·C ...wobble pairs form near physiological pH and have relatively minor effects on helix geometry, making them especially important in biology. Herein, we investigate effects of helix position, temperature, and ionic strength on pK a shifting in A+·C wobble pairs in DNA. We observe that pK a shifting is favored by internal A+·C wobbles, which have low cooperativities of folding and make large contributions to stability, and disfavored by external A+·C wobbles, which have high folding cooperativities but make small contributions to stability. An inverse relationship between pK a shifting and temperature is also found, which supports a model in which protonation is enthalpically favored overall and entropically correlated with cooperativity of folding. We also observe greater pK a shifts as the ionic strength decreases, consistent with anticooperativity between proton binding and counterion-condensed monovalent cation. Under the most favorable temperature and ionic strength conditions tested, a pK a of 8.0 is observed for the A+·C wobble pair, which represents an especially large shift (∼4.5 pK a units) from the unperturbed pK a value of adenosine. This study suggests that protonated A+·C wobble pairs exist in DNA under biologically relevant conditions, where they can drive conformational changes and affect replication and transcription fidelity.
Small ribozymes such as the hairpin, hammerhead, VS, glm S, and hepatitis delta virus (HDV) are self-cleaving RNAs that are typically characterized by kinetics and structural methods. Working with ...these RNAs requires attention to numerous experimental details. In this chapter we focus on four different experimental aspects of ribozyme studies: preparing the RNA, mapping its structure with reverse transcription and end-labeled techniques, solvent isotope experiments, and co-transcriptional cleavage assays. Although the focus of these methods is the HDV ribozyme, the methods should be applicable to other ribozymes.
SCFE occurs in 10 per 100,000 in some regions of the United States with the incidence continuing to increase. Percutaneous screw fixation is a well-accepted treatment for this disorder for over 20 ...years but management of complications is not well elucidated in the literature.
We describe a case where a traumatic unstable SCFE that was initially treated with closed reduction and fixation with a single transphyseal screw went on to hardware failure with recurrence of the deformity. The complication was successfully treated with closed reduction and re-cannulating the fractured screw within the epiphysis and extracting it using a conical extraction screw commonly referred to as an "easy out." Three trans physeal screws were then placed for improved fixation strength. Follow-up at 9 months demonstrates a fused physis and no signs of avascular necrosis of the femoral head.
Percutaneous management of SCFE screw breakage is possible utilizing specialized instruments and a precise and gentle manipulation preventing the need for more invasive treatments with their obligatory potential complications profile.
The ability of RNA to both store genetic information and catalyze chemical reactions has led to the RNA world hypothesis, wherein RNA served as the primary biological molecule. In comparison to the ...variety of chemical side chains available to proteins, RNA (which bears only four, chemically similar side chains) seems to be woefully inadequate, so much so that the discovery of catalytic RNAs in 1982 came as a surprise to many and created a whole new field of study. Since then, a steady stream of RNA-related discoveries have had profound impact on the fields of chemistry, biochemistry, genetics, and molecular biology. One particularly powerful catalytic strategy utilized by protein enzymes is general acid-base catalysis. Proteins can utilize amino acid side chains like histidine and lysine which have pKa values at or above physiological pH (∼7), to attain efficient proton transfer or electrostatic catalysis, respectively. Nucleic acid bases, on the other hand, have no ionizing groups with pKas in this range and, to make matters worse, routinely experience pK a shifts further from this ideal range upon standard helix formation because the standard protonation state is required for Watson-Crick base pairing. On the other hand, helix formation of non-Watson Crick pairs that require non-standard protonation states causes a pK a shift towards neutrality (or beyond). We hypothesized that coupling many interactions to a protonation event through cooperative structure formation could shift nucleobases pKa values even further. I designed model systems to study folding cooperativity in RNA and DNA hairpins as a function of helical context. Significant cooperativity was observed at the terminus of a helix; in fact, it is demonstrated that a terminal base pair will not form in the absence of a penultimate base pair in both RNA and DNA hairpins. I also note that the thermodynamic impact of mutations at the terminus are small, particularly when compared to the same mutations performed on bases in the middle of the helix. It is at this point where RNA and DNA trends differ: DNA does not exhibit strong cooperativity in the middle of a helix, while RNA does. This difference is attributed to differences in helical structure and flexibility. Experiments were then performed to determine the molecular basis for an unexpectedly large structural disruption observed in the study mentioned above. It was demonstrated that unsatisfied hydrogen bond acceptors can play a surprising and major role in determining the stability (and therefore specificity) of base pair formation in the RNA bases studied. An example of the impact of these interactions is provided in tRNA folding specificity, wherein removing an unsatisfied hydrogen bond acceptor using a base found in the anticodon (2-thiocytosine) increases specificity for canonical Watson Crick base pairing. Having obtained interesting structural information, we turned to pH studies, beginning with a study that details sources of error in experimental pH measurements. This study was instigated by collaboration with Dr. Andrea Cerrone-Szakal on hepatitis delta virus (HDV) ribozyme kinetics in high salt, with an ultimate desire examine pKa shifting under in vivo-like conditions of unusual salt and crowding. A substantial ion-specific effect on pH measurements was found, causing meter readings that are almost 1 unit too low, particularly at high ionic strengths. It was determined that the ionic strength of a solution will affect not only the pK a of the solution species themselves, but also the ability of a pH electrode to produce an accurate reading. A two-step calibration method is presented to correct for this error, and this method is applied to a study of the p Ka of C in a single stranded RNA (UUCUU) representing the unfolded state. Lastly, experiments are presented that examine the folded-state p Ka of a dsDNA model system. Effects of context, temperature, and ionic strength on the pKa of a noncanonical A+·C wobble pair are presented. This pair requires a protonated A to form, and hence meets the aforementioned criteria for p Ka shifting; furthermore, by applying knowledge gained in the cooperativity studies, the effect of cooperativity on pK a shift is studied as well. Conditions under which very large p Ka shifts can be observed are identified, and p Ka values at and beyond neutrality for this pair are reported, an observation that supports the potential for both general acid-base and electrostatic catalysis in nucleic acids. We present significant p Ka shifts in internally located base pairs only, suggesting that the folding free energy, rather than folding cooperativity, is the most important predictor of pKa shifting. (Abstract shortened by UMI.)
The recent commercialization of unoccupied aerial vehicles (UAVs) has facilitated their incorporation into a variety of ecological studies. While UAVs are able to provide accurate visual data of ...marine species from an aerial perspective, these devices have some limitations that make measuring marine animals below the surface challenging. Many marine organisms are often visible from the air, but are deeper in the water column, and current methods cannot measure animals below the surface. Here, we developed and tested a stereo-video camera (SVC) system that was mounted onto a commercially-available UAV. We used the SVC-UAV to conduct remote body-size measurements for two marine species: the green sea turtle (Chelonia mydas) and the nurse shark (Ginglymostoma cirratum). When comparing SVC measurements to those taken by hand, the SVC-UAV had a mean absolute error (MAE) of 4.44 cm (n = 6; mean percent error (MPE) = 10.6%) for green sea turtles and 7.16 cm absolute error (n = 1; PE = 3.6%) for the nurse shark. Using a linear model, we estimated the slope of the SVC versus hand measurements for green sea turtles to be 1.085 (±0.099 SE), and accounting for the standard error, a measurement bias was not apparent. Using model selection, based on a global model predicting MAE from animal distance to the SVC and body size, the top ranked model was the intercept-only model. This indicates that neither animal distance nor body size strongly influenced measurement error. Incorporating SVC systems into UAVs can allow for relatively accurate measurements of near surface-dwelling marine species. To our knowledge, there is no other stand-alone SVC for UAVs available that offers similar accuracy and utility.
Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of ...the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (
, rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of
(Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.
Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available ...sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.
We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.
Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (http://www.ncbi.nlm.nih.gov/sra?term=SRP002610). Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html. Seven Additional files are available..
The recent commercialization of unoccupied aerial vehicles (UAVs) has facilitated their incorporation into a variety of ecological studies. While UAVs are able to provide accurate visual data of ...marine species from an aerial perspective, these devices have some limitations that make measuring marine animals below the surface challenging. Many marine organisms are often visible from the air, but are deeper in the water column, and current methods cannot measure animals below the surface. Here, we developed and tested a stereo-video camera (SVC) system that was mounted onto a commercially-available UAV. We used the SVC-UAV to conduct remote body-size measurements for two marine species: the green sea turtle (Chelonia mydas) and the nurse shark (Ginglymostoma cirratum). When comparing SVC measurements to those taken by hand, the SVC-UAV had a mean absolute error (MAE) of 4.44 cm (n = 6; mean percent error (MPE) = 10.6%) for green sea turtles and 7.16 cm absolute error (n = 1; PE = 3.6%) for the nurse shark. Using a linear model, we estimated the slope of the SVC versus hand measurements for green sea turtles to be 1.085 (±0.099 SE), and accounting for the standard error, a measurement bias was not apparent. Using model selection, based on a global model predicting MAE from animal distance to the SVC and body size, the top ranked model was the intercept-only model. This indicates that neither animal distance nor body size strongly influenced measurement error. Incorporating SVC systems into UAVs can allow for relatively accurate measurements of near surface-dwelling marine species. To our knowledge, there is no other stand-alone SVC for UAVs available that offers similar accuracy and utility.
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