Showing papers by "Jennifer M. Heemstra published in 2021"

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors

Abstract: Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

Quantifying fear of failure in STEM: modifying and evaluating the Performance Failure Appraisal Inventory (PFAI) for use with STEM undergraduates

Abstract: The ability to navigate obstacles and embrace iteration following failure is a hallmark of a scientific disposition and is hypothesized to increase students’ persistence in science, technology, engineering, and mathematics (STEM). However, this ability is often not explicitly explored or addressed by STEM instructors. Recent collective interest brought together STEM instructors, psychologists, and education researchers through the National Science Foundation (NSF) research collaborative Factors affecting Learning, Attitudes, and Mindsets in Education network (FLAMEnet) to investigate intrapersonal elements (e.g., individual differences, affect, motivation) that may influence students’ STEM persistence. One such element is fear of failure (FF), a complex interplay of emotion and cognition occurring when a student believes they may not be able to meet the needs of an achievement context. A validated measure for assessing FF, the Performance Failure Appraisal Inventory (PFAI) exists in the psychological literature. However, this measure was validated in community, athletic, and general undergraduate samples, which may not accurately reflect the motivations, experiences, and diversity of undergraduate STEM students. Given the potential role of FF in STEM student persistence and motivation, we felt it important to determine if this measure accurately assessed FF for STEM undergraduates, and if not, how we could improve upon or adapt it for this purpose. Using exploratory and confirmatory factor analysis and cognitive interviews, we re-validated the PFAI with a sample of undergraduates enrolled in STEM courses, primarily introductory biology and chemistry. Results indicate that a modified 15-item four-factor structure is more appropriate for assessing levels of FF in STEM students, particularly among those from groups underrepresented in STEM. In addition to presenting an alternate factor structure, our data suggest that using the original form of the PFAI measure may significantly misrepresent levels of FF in the STEM context. This paper details our collaborative validation process and discusses implications of the results for choosing, using, and interpreting psychological assessment tools within STEM undergraduate populations.

Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics.

Abstract: Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin–Watson–Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.

Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay

Abstract: Adenosine-to-inosine (A-to-I) editing is a conserved RNA modification that contributes to immune response and overall cellular function. RNA editing patterns can vary significantly between different cell and tissue types, and hyperactive A-to-I signatures are indicative of several diseases, including cancer and autoimmune disorders. Because of the importance of these differences, there is significant need for efficient methods to measure overall A-to-I editing levels in cellular RNA. The current standard approach relies on RNA-seq to indirectly detect editing sites, which requires large investments in time and material as well as extensive computational analysis. Here, we utilize Endonuclease V (EndoV), which binds to inosine in RNA, to develop a protein-based chemiluminescent bioassay to directly profile A-to-I editing activity. We previously showed that EndoV can enrich edited transcripts prior to RNA-seq, and we now leverage this activity to construct an EndoV-linked immunosorbency assay (EndoVLISA) as a rapid chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimize and validate our assay, illustrating highly selective and sensitive detection of inosine in RNA. We then demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current standard RNA-seq approaches. Lastly, we deploy our EndoVLISA for profiling differential A-to-I RNA editing signatures in normal and diseased human tissue, illustrating the utility of our platform as a diagnostic bioassay. Together, the EndoVLISA method is cost-effective, straightforward, and utilizes common laboratory equipment, offering a highly accessible new approach for studying A-to-I editing. Moreover, the multi-well plate format makes this the first assay amenable for direct high-throughput quantification of A-to-I editing for applications in disease detection and drug development.