Exploring DNA-protein interactions on the single DNA molecule level using nanofluidic tools

DNA-protein interactions are at the core of the cellular machinery and single molecule methods have revolutionized the possibilities to study, and our understanding of these interactions on the molecular level. Nanofluidic channels have been extensively used for studying single DNA molecules during the last twelve years and in this review, we discuss how this experimental platform has been extended to studies of DNA-protein interactions. We first present how the design of the device can be tailored for the specific DNA-protein system studied and how the channels can be passivated to avoid non-specific binding of proteins. We then focus on describing the different kinds of DNA-interacting proteins that have been studied in nanofluidic devices, including proteins that compact DNA and proteins that form filaments on DNA. Our main objective is to highlight the diverse functionalitiesof DNA-protein systems that have been characterized using nanofluidic structures and hence demonstrate the versatility of these experimental tools. We finally discuss potential future directions studies of DNA-protein complexes in nanochannels might take, including specific DNA-protein systems that are difficult to analyze with traditional techniques, devices with increased complexity, and fully integrated lab-on-a-chip devices for analysis of material extracted from (single) cells

Heidegger and the Problem of Phenomenality

This PhD thesis is an extended critical investigation of Martin Heidegger s influential account of the problem of phenomenality, i.e., of how things show up as meaningful phenomena in our experience. As such, it is also a study of his effort to develop and probe the question of phenomenology, i.e., what it means to see, understand, and articulate such phenomena. The aim of the thesis is both historical and systematic. On the one hand, it offers a unified interpretation of how Heidegger s struggle with the problem of phenomenality unfolds during the main stages of his philosophical development, from the early Freiburg lecture courses 1919-1923, over the Marburg years and the publication of Being and Time in 1927, up to his later thinking stretching from the mid 1930s to the early 1970s. It is argued that the problem of phenomenality constitutes one of the core problems that Heidegger is concerned with from beginning to end, and that focusing on this problem allows us to shed new light on the philosophical logic and motives behind the main changes that his thinking undergoes along the way. On the other hand, the thesis examines both the philosophical power and the problems and ambiguities of Heidegger s consecutive attempts to account for the structure and dynamics of phenomenality. In particular, it critically interrogates Heidegger's basic idea that our experience of meaningful phenomena is determined by our prior understanding of the historical contexts of meaning in which we always already live. A central argument of the thesis is that Heidegger s conception of the historical structure of phenomenality raises the decisive question of how to distinguish between historical prejudice and primordial understanding, and that Heidegger s inability to answer this question in Being and Time generates a deep ambiguity between his program of historical-destructive thinking and his employment of a Husserlian intuition-based phenomenological method in his concrete investigation. Moreover, it is argued that Heidegger s later thinking of the clearing/event of being is centrally motivated by the effort to answer precisely this question by showing how a historical world can arise and give itself as a binding destiny. Ultimately, however, the thesis suggests elaborating on the criticisms previously presented by, e.g., Ernst Tugendhat and Emmanuel Levinas that Heidegger s radical historicization of phenomenality makes him unable to account either for the truth of our understanding or for the ethical-existential significance of other persons.Den här avhandlingen är en studie i Martin Heideggers filosofi med särskilt fokus på hans försök att komma till rätta med fenomenalitetens problem, det vill säga med frågan hur saker och ting kan vara givna och visa sig som meningsfulla fenomen i vår erfarenhet. I avhandlingen undersöker och diskuterar jag också Heideggers syn på fenomenologin, alltså på vad det vill säga att se, förstå och artikulera våra erfarenheters mening. Med fenomenalitetens problem som ledtråd utvecklar avhandlingen en utförlig tolkning av de centrala faserna av Heideggers filosofiska tankeväg: del 1 behandlar Heideggers tidiga Freiburg-föreläsningarna 1919-1923; del 2 fokuserar på hans tänkande under Marburg-åren och i det tidiga huvudverket Vara och tid från 1927; del 3 behandlar Heideggers senare tänkande från mitten av 1930-talet till början 1970-talet. Jag argumenterar för att fenomenalitetens problem står i centrum för Heideggers filosofiska intresse från början till slut och att vi genom att följa hans kamp med denna problematik kan kasta nytt ljus över hans filosofiska utveckling och belysa de motiv och problem som driver hans tänkande framåt. Avhandlingen är dock inte bara en historisk utläggning. Ambitionen är också att kritiskt granska och avgränsa den filosofiska halten i Heideggers tänkande. Avhandlingens kritiska frågande riktar sig i synnerhet mot Heideggers grundtanke att det bara är på basen av vår förförståelse av de historiska meningskontexter som vi alltid redan lever i som vi kan erfara och förstå entiteter som meningsfulla fenomen som stolar, träd, människor, etc. Idén om fenomenalitetens historiska struktur väcker svåra frågor. Till exempel: Hur ska vi kunna skilja mellan historisk fördom och sann förståelse om vi inte har tillgång till meningskällor bortom våra historiska meningssammanhang? Kan våra historiska meningskontexter inklusive våra historiska värderingar och normer verkligen vara ursprunget till den etisk-existentiella betydelse människor och saker har för oss? Heidegger har stora problem att hantera dessa frågor, vilket ger upphov till djupa ambivalenser och brister i hans tänkande. I sista hand argumenterar jag för att Heideggers radikala historisering av vår erfarenhet och förståelse gör att han inte kan redogöra för vår förståelses sanning och osanning. Hans försök att härleda all betydelse ur historien bygger också på ett grundläggande förnekande av det sätt på vilket andra människor möter oss som etiskt-existentiellt betydelsefulla och angelägna oavsett vilken historisk kontext vi lever i

Single-Molecule Trapping and Measurement in a Nanostructured Lipid Bilayer System

The repulsive electrostatic force between a biomolecule and a like-charged surface can be geometrically tailored to create spatial traps for charged molecules in solution. Using a parallel-plate system composed of silicon dioxide surfaces, we recently demonstrated single-molecule trapping and high precision molecular charge measurements in a nanostructured free energy landscape. Here we show that surfaces coated with charged lipid bilayers provide a system with tunable surface properties for molecular electrometry experiments. Working with molecular species whose effective charge and geometry are well-defined, we demonstrate the ability to quantitatively probe the electrical charge density of a supported lipid bilayer. Our findings indicate that the fraction of charged lipids in nanoslit lipid bilayers can be significantly different from that in the precursor lipid mixtures used to generate them. We also explore the temporal stability of bilayer properties in nanofluidic systems. Beyond their relevance in molecular measurement, such experimental systems offer the opportunity to examine lipid bilayer formation and wetting dynamics on nanostructured surfaces

Real-time compaction of nanoconfined DNA by an intrinsically disordered macromolecular counterion

We demonstrate how a recently developed nanofluidic device can be used to study protein-induced compaction of genome-length DNA freely suspended in solution. The protein we use in this study is the hepatitis C virus core protein (HCVcp), which is a positively charged, intrinsically disordered protein. Using nanofluidic devices in combination with fluorescence microscopy, we observe that protein-induced compaction preferentially begins at the ends of linear DNA. This observation would be difficult to make with many other single-molecule techniques, which generally require the DNA ends to be anchored to a substrate. We also demonstrate that this protein-induced compaction is reversible and can be dynamically modulated by exposing the confined DNA molecules to solutions containing either HCVcp (to promote compaction) or Proteinase K (to disassemble the compact nucleo-protein complex). Although the natural binding partner for HCVcp is genomic viral RNA, the general biophysical principles governing protein-induced compaction of DNA are likely relevant for a broad range of nucleic acid-binding proteins and their targets

C-terminal truncation of α-synuclein alters DNA structure from extension to compaction

Parkinson\u27s disease (PD) is linked to aggregation of the protein α-synuclein (aS) into amyloid fibers. aS is proposed to regulate synaptic activity and may also play a role in gene regulation via interaction with DNA in the cell nucleus. Here, we address the role of the negatively-charged C-terminus in the interaction between aS and DNA using single-molecule techniques. Using nanofluidic channels, we demonstrate that truncation of the C-terminus of aS induces differential effects on DNA depending on the extent of the truncation. The DNA extension increases for full-length aS and the (1–119)aS variant, but decreases about 25% upon binding to the (1–97)aS variant. Atomic force microscopy imaging showed full protein coverage of the DNA at high aS concentration. The characterization of biophysical properties of DNA when in complex with aS variants may provide important insights into the role of such interactions in PD, especially since C-terminal aS truncations have been found in clinical samples from PD patients

Combining dense and sparse labeling in optical DNA mapping

Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids

A nanofluidic device for real-time visualization of DNA–protein interactions on the single DNA molecule level

Single DNA molecule techniques have revolutionized our understanding of DNA-protein interactions. Traditional techniques for such studies have the major drawback that the DNA molecule studied is attached to a bead or a surface. Stretching of DNA molecules in nanofluidic channels has enabled single-molecule studies of DNA-protein interactions without the need of tethering the molecule to a foreign entity. This in turn allows for studying reactions along the whole extension of the molecule, including the free DNA ends. However, existing studies either rely on measurements where all components are mixed before introduction into the nanochannels or where passive diffusion brings the reagents to the confined DNA molecule. We here present a new generation of nanofluidic devices, where active exchange of the local environment within the nanofluidic channel is possible, while keeping the DNA molecule stretched and in confinement. To demonstrate the functionality of this novel device we added different analytes, such as SDS, spermidine and DNase I, to YOYO-1 stained DNA and studied the response in real time. We also performed a FRET-based reaction, where two different analytes were added sequentially to the same DNA molecule. We believe that this design will enable in situ mapping of complex biochemical processes, involving multiple proteins and cofactors, on single DNA molecules as well as other biomacromolecules

Complex conformational dynamics of the heart failure‐associated pre‐mirna‐377 hairpin revealed by single‐molecule optical tweezers

pre‐miRNA‐377 is a hairpin‐shaped regulatory RNA associated with heart failure. Here, we use single‐molecule optical tweezers to unzip pre‐miRNA‐377 and study its stability and dy-namics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabi-lize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nu-cleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre‐miRNA‐377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA‐377 hairpin and perturbs its folding. The knowledge regarding pre‐miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment