We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid’s structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Background: Canadian Blood Services (CBS) screens donors based on group status (e.g., men who have sex with men, MSM) instead of specific, high-risk sexual practices (e.g., occurrence of condomless sex). The MSM screening question is embedded in a cluster of questions about stigmatized attributes such as history of imprisonment and illicit substance use. This juxtaposition of the "MSM question" and stigmatized attributes may unintentionally cause blood donors to perceive MSM more negatively. The aim of this research is to determine whether the CBS donor eligibility questionnaire generates negative bias against MSM.Study Design and Methods: A national, randomized online study of 903 CBS donors was conducted. Participants completed either the existing blood donor eligibility questionnaire or a modified donor questionnaire that repositioned the MSM question among neutral questions. After completing the existing or modified questionnaire, bias against MSM was measured using the sexuality implicit association test (IAT) and Modern Homonegativity Scale -Gay Men (MHS-G). Lastly, participants estimated prevalence rates among MSM of certain stigmatized behaviors.Results: Participants who completed the existing donor eligibility questionnaire more strongly associated gay men with negative attributes on the IAT (p one-tailed = .045), suggesting question position generated implicit negative bias toward MSM. Responses to the MHS-G (p one-tailed = .506) and prevalence estimation task (p = .443) indicated that question order had no significant impact on explicit bias.Discussion: Positioning the MSM screening question among stigmatizing questions creates implicit negative bias against MSM. Policy makers should be mindful of question positioning when designing donor questionnaires.

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

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