The magnetic tweezer is a single molecule instrument that can measure the extension of biomolecules by tracking tethered probe particles. Due to its experimental simplicity and ability to apply a constant external force to probe particles, the magnetic tweezer is ideally suited to measuring single molecule dynamics under applied tension. In this thesis, I push the resolution of magnetic tweezers to angstrom spatial resolution by using a high-speed camera which can achieve more than 10 kHz temporal resolution.

I investigate the fundamental limits to magnetic tweezer resolution from basic principles, and investigate different types of light coherence to arrive on a new choice of light source for the magnetic tweezer: the superluminescent diode. I explore Langevin dynamics of tethered probe particles using the Allan variance, a more intuitive take on thermal fluctuations than the Power Spectral Density. I push the achievable resolution limits by using smaller beads, introduce higher gradient magnets to apply biologically relevant forces on these smaller beads, and introduce a Fourier filter system to achieve higher spatial resolution.

Intrinsically disordered proteins (IDPs) do not adopt a stable three-dimensional structure, thus posing a challenge to common characterization techniques that have found success with folded proteins. While IDP conformational characterization has been explored with small angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET), there is a demand for alternative experimental techniques that can provide new perspectives in this field.

Beyond biological IDPs, quantification of rapidly fluctuating conformational states is also crucial for the design and development of synthetic sequence-defined polymers. Inspired by the ability of proteins to assemble structures with high specificity, a class of polymeric materials emerged whose sequences can be tailored to achieve the same level of complexity. Among these, polypeptoids are the most promising due to their precise synthesis and chemical modifiability. The rational design of polypeptoid materials calls for a fundamental understanding of how their sequences influence their conformational behaviors.

We approach the conformational study of these two polymer systems through entropic elasticity measurements using magnetic tweezers. Force-extension measurements reveal insights into polymer structure at a broad range of length scales, from the local stiffness described by the persistence length to long-range interactions quantified by the Flory exponent. As these sequence-defined polymers are typically too short for magnetic tweezers experiments, we also explore ligation schemes to form long tether constructs. Here, we describe the investigation of the conformations of a model IDP, the neurofilament low molecular weight tail region (NFLt). Despite the high charge content, we find that NFLt is more compact than expected due to local hydrophobic attractions. Our results, specifically Flory exponent measurements in denaturant concentrations, are in remarkable agreement with previous results on other IDPs using SAXS or FRET. We thus demonstrate a novel framework to characterize the conformational ensembles of IDPs through entropic elasticity analysis.

We also investigate the effects of charge spacing on polypeptoid conformation through simple sequence designs and find conflicting observations on the electrostatic influence. Whereas the local flexibility is unperturbed, the solubility behavior in increasing ionic strength is dependent on the charge spacing. We attribute this unique conformational behavior to the chemical structure of polypeptoids, whose charge groups are on side chains. Our results contribute novel insights into the sequence-conformation relationship, as well as raising new questions on polyelectrolyte physics.

Through the studies on NFLt and charged polypeptoids, we establish a previously unexplored approach of using magnetic tweezers experiments and entropic elasticity analysis to quantify the conformations of sequence-defined polymers. For IDPs, magnetic tweezers can complement existing techniques (SAXS and FRET) and advance the general knowledge of protein conformational disorder. For polypeptoids, this framework can be applied for the investigation of other sequence effects, such as ampholyticity or hydrophobicity.

DNA organization within a cell is multifaceted and dynamic. Not only does chromatin structure vary with position, as there is significant heterogeneity in the degree of local compaction within the global DNA complex, but it also changes with time, showing dependence on, e.g., the growth stage of a cell. The condensation state of DNA is intimately coupled to gene accessibility and expression: changes to DNA organization can modulate transcription and, in turn, transcribed RNA can reorganize chromatin structure. We sought to develop a more tractable, in vitro platform to probe this complex relationship and utilized a self-assembling DNA nanostar (NS) as the principal component of a chromatin mimic. In this dissertation, I will discuss our research with condensed phases of self-assembled NSs, describing structural properties of the system's two phases (i.e. NS-networks & NS-liquids), features of NS-liquids that enable application as a model of membraneless organelles, and initial work examining the transcription of genes integrated within NS-liquids.

Conservative estimates indicate that 30% of known proteins contain long (>40 residues) intrinsically disordered regions (IDRs). In contrast to globular proteins, IDRs adopt multiple distinct conformations in their native state, similar to a random-walk polymer in a good solvent. While the sequence properties of IDRs have been extensively studied, their physical properties are still poorly understood. Characterizing these physical properties is an important step towards understanding the numerous biological functions and diseases associated with IDRs. Recently, Magnetic Tweezers (MTs) have emerged as a powerful tool for determining the structural properties of polymers in a manner that is orthogonal to other approaches, e.g., scattering experiments. With MTs, researchers use the thermodynamic effects of applied tension to study the conformations of single polymers via their end-to-end extension. This approach is particularly desirable for IDRs because many of them are found in the cellular cytoskeleton, where they play a critical structural and mechanical role. In the first part of this dissertation, I present my work on improving the MT technique with the aim of studying IDRs. I develop a computational tool for the robust calibration of forces and their uncertainty, an important but often overlooked aspect of these experiments. I also study the effects of surfaces on the low-force entropic elastic response of polymers, showing that they can be used to extract the radius of gyration. In the second part of this dissertation, I present the unexpected finding of glassy dynamics in a model IDR system, a polyprotein of the disordered neurofilament light tail (NFLt) domain. The NFLt is part of a large group of IDRs in neurons that are responsible for the structure and mechanics of the axon. Glassy dynamics in globular proteins was a major finding nearly 50 years ago that emphasized the importance of protein dynamics. However, it is attributed to conformational behaviors that are missing from IDRs. Nevertheless, using MT experiments, I show that a NFLt polyprotein’s extension changes, in response to a change in applied tension, with a nonexponential time dependence that is history dependent, two characteristic features of glassy systems. I show that the extension changes can be predicted using a phenomenological framework adapted from bulk glassy systems. Finally, I show that the glassy dynamics can be understood in terms of multiple, independent, and heterogeneous globules within a single NFLt. This mechanism for glassy dynamics is novel and likely to apply broadly to other IDRs.

The biopolymer hyaluronic acid (HA) is ubiquitous in extracellular spaces and forms complexes with proteins and proteoglycans that contribute to tissue mechanical properties, provide lubrication at biointerfaces, and affect cell adhesion and motility. It is not well-understood how structural features within single HA chains control its biological roles and its potential for use in biomaterials. HA's structural length scales (persistence length, charge spacing) are intermediate relative to other biopolymers; this makes it a useful model system for studying polyelectrolyte physics. We use magnetic tweezers to investigate how salt and HA-binding ligands can modify its random coil structure.

We present data on HA's behavior in mixed valence salt solutions, and find a regime of conformational insensitivity to ionic strength. Experiments on another flexible charged biopolymer, single-stranded DNA, confirm the effect is a general electrostatic phenomenon. With simulations and theory, we build a picture of how ion exchange within a counterion-rich layer near the chain counteracts changes in the bulk ionic strength. We also use magnetic tweezers to measure changes in HA structure associated with the formation of HA-proteoglycan bottlebrush complexes. As the large proteoglycan aggrecan binds, it induces tension in the central HA chain. Using established bottlebrush theory, we connect this tension to structural features of aggrecan. Overall, we find magnetic tweezers are sensitive to HA's random coil structure and its modification by solute interactions.

It is known about the role of technological factors in HR. Technology changes the world of business and transforms the labor market. This work focuses in particular on the impact of new technologies to provide employment to workers, as well as self-employment. There are clear possibilities and wider use of digital tools. The government, companies and individuals today can benefit greatly from new "digital jobs" and from the use of digital tools. However, technology also brings risks. Some jobs can be digitized to varying degrees, and some workers or part of their functions are replaced with new technology. The ability to take advantage of these opportunities will vary from individual to individual; workers with higher skill levels are more likely to benefit, while those with lower levels of skills may be less willing to private new technologies, and therefore may be more at risk of poorer quality of work and even loss of work. Moreover, it seems that the larger the technology gap between domestic and foreign establishments.

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