Controlling Assembly and Activity of Biomimetic DNA Nanopores

Biological channels control the transport of vital biomolecular cargo across the cellular membrane. Reflecting the channels’ critical role, replicating their structure and improving on their function is of significant biomedical and scientific interest. However, de novo design of pores with the typical biological building material of polypeptides is challenging. DNA, by contrast, offers unrivalled structural control due to the simplicity and specificity of Watson-Crick base-pairing. Taking advantage of these properties, DNA membrane pores have been rationally designed with tuneable dimensions. The overall aim of this thesis is to advance on the simple barrel-like synthetic pores and create higher-order function to control pore formation and transport by means of exogenous triggers. The first aim of this thesis was the development of a model system to probe DNA hybridisation under steric constraints. By exploring the effect of DNA hybridisation under steric constraints, such as at membranes and on DNA nanostructures, greater insight was provided for the design of DNA nanopore that assemble in situ and respond to exogenous triggers. The second aim was to design a DNA nanopore that would mimic protein pore formation by undergoing triggered assembly. To transition the inactive pre-pore monomers to an active membrane-spanning oligomeric pore, the locked monomers can be unlocked in the presence of keys to trigger pore assembly. The pore advances functional DNA nanotechnology and synthetic biology by imparting targeted selectivity for pore activity and by serving as a synthetic mimic. The third aim was to build a DNA nanopore that functions as a synthetic protein-gate, allowing the transport of molecular cargo only in the presence of a target exogenous trigger. To function as a protein-gate, a DNA nanopore was designed with a lid featuring an aptamer sequence. In the rational designed structure, the binding of a target protein to the aptamer actuates the lid to open the pore into a transport-active state. A pore with such selective control could then be used in targeted drugdelivery

Age measurements of the Precambrian rocks of the Death Valley-Mojave Desert region, California

Ar^(40)-K^(40) and Sr^(87)-R^(87) age measurements in the eastern Mojave Desert indicate two separate early Precambrian events (see table). The older event is approximately 1650 m.y. old and is evidenced by pegmatites and associated metamorphic rocks in the Mountain Pass district. Ages were measured on coarse muscovite and potassium feldspar, MP-1 and MP-2, from a pegmatite which cuts across biotite-bearing gneisses, MP-7 and MP-9. These data confirm the widespread areal extent of this ancient metamorphic terrane. Ages of biotite, MP-21 and MP-22, from the shonkinite, which intrudes the metamorphic rocks, at Mountain Pass and the Rb-Sr age of potassium feldspar, MM-3f, from granite in the Marble Mountains suggest a period of igneous intrusion in the 1350 to 1410 m.y. interval. Metamorphic rocks in the central Panamint Range have been mapped and are shown to be stratigraphically early Precambrian. K-Ar ages of approximately 80 m.y. have been measured on biotite, muscovite, and hornblende. The minerals show no memory of a Precambrian age. The early Precambrian rocks show no evidence of a younger period of metamorphism. However, a younger metamorphism can be recognized in the overlying Precambrian(?) Noonday dolomite and Johnnie formation

Migration of radiogenic strontium during metamorphism

Study of the concentration and isotopic composition of strontium and rubidium in hornblende diorite dikes and sills of Precambrian age from the Panamint Mountains of California showed that these rock systems were enriched in radiogenic Sr during a late Mesozoic metamorphism. Enrichments in radiogenic Sr were observed for total-rock samples which yielded apparent ages of up to 34,000 m.y., although there is no obvious petrographic evidence for such metasomatic changes. These results indicate that some caution is necessary in interpreting the Sr isotopic composition of rocks in terms of their original source or in assuming that ‘total rocks’ form closed systems. In general, the initial isotopic Sr composition should be determined, not assumed

Correlation of Ordovician and Silurian Formations of Eastern Montana

Correlation charts made from electric log data show the relationship of Ordovician and Silurian strata in eastern Montana. The Silurian sediments terminate westward in eastern Montana, but the Ordovician sediments are present westward into central Montana

DNA Nanodevices with Selective Immune Cell Interaction and Function

DNA nanotechnology produces precision nanostructures of defined chemistry. Expanding their use in biomedicine requires designed biomolecular interaction and function. Of topical interest are DNA nanostructures that function as vaccines with potential advantages over nonstructured nucleic acids in terms of serum stability and selective interaction with human immune cells. Here, we describe how compact DNA nanobarrels bind with a 400-fold selectivity via membrane anchors to white blood immune cells over erythrocytes, without affecting cell viability. The selectivity is based on the preference of the cholesterol lipid anchor for the more fluid immune cell membranes compared to the lower membrane fluidity of erythrocytes. Compacting DNA into the nanostructures gives rise to increased serum stability. The DNA barrels furthermore functionally modulate white blood cells by suppressing the immune response to pro-inflammatory endotoxin lipopolysaccharide. This is likely due to electrostatic or steric blocking of toll-like receptors on white blood cells. Our findings on immune cell-specific DNA nanostructures may be applied for vaccine development, immunomodulatory therapy to suppress septic shock, or the targeting of bioactive substances to immune cells

Structure and dynamics of an archetypal DNA nanoarchitecture revealed via cryo-EM and molecular dynamics simulations

DNA can be folded into rationally designed, unique, and functional materials. To fully realise the potential of these DNA materials, a fundamental understanding of their structure and dynamics is necessary, both in simple solvents as well as more complex and diverse anisotropic environments. Here we analyse an archetypal six-duplex DNA nanoarchitecture with single-particle cryo-electron microscopy and molecular dynamics simulations in solvents of tunable ionic strength and within the anisotropic environment of biological membranes. Outside lipid bilayers, the six-duplex bundle lacks the designed symmetrical barrel-type architecture. Rather, duplexes are arranged in non-hexagonal fashion and are disorted to form a wider, less elongated structure. Insertion into lipid membranes, however, restores the anticipated barrel shape due to lateral duplex compression by the bilayer. The salt concentration has a drastic impact on the stability of the inserted barrel-shaped DNA nanopore given the tunable electrostatic repulsion between the negatively charged duplexes. By synergistically combining experiments and simulations, we increase fundamental understanding into the environment-dependent structural dynamics of a widely used nanoarchitecture. This insight will pave the way for future engineering and biosensing applications

Triggered Assembly of a DNA-Based Membrane Channel

Chemistry is in a powerful position to synthetically replicate biomolecular structures. Adding functional complexity is key to increase the biomimetics' value for science and technology yet is difficult to achieve with poorly controlled building materials. Here, we use defined DNA blocks to rationally design a triggerable synthetic nanopore that integrates multiple functions of biological membrane proteins. Soluble triggers bind via molecular recognition to the nanopore components changing their structure and membrane position, which controls the assembly into a defined channel for efficient transmembrane cargo transport. Using ensemble, single-molecule, and simulation analysis, our activatable pore provides insight into the kinetics and structural dynamics of DNA assembly at the membrane interface. The triggered channel advances functional DNA nanotechnology and synthetic biology and will guide the design of controlled nanodevices for sensing, cell biological research, and drug delivery

Design, assembly, and characterization of membrane-spanning DNA nanopores

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores’ chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore–membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2–3 weeks for DNA origami pores

The effect of aging on the autophagic and heat shock response in human peripheral blood mononuclear cells

Autophagy is a lysosome degradation pathway through which damaged organelles and macromolecules are degraded within the cell. A decrease in activity of the autophagic process has been linked to several age-associated pathologies, including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction. Here, we examined the differences in the autophagic response using autophagy-inducer rapamycin (Rapa) in peripheral blood mononuclear cells (PBMCs) from young (21.8 ± 1.9 years) and old (64.0 ± 3.7 years) individuals. Furthermore, we tested the interplay between the heat shock response and autophagy systems. Our results showed a significant increase in LC3-II protein expression in response to Rapa treatment in young but not in old individuals. This was associated with a decreased response in MAP1LC3B mRNA levels, but not SQSTM1/p62. Furthermore, HSPA1A mRNA was upregulated only in young individuals, despite no differences in HSP70 protein expression. The combined findings suggest a suppressed autophagic response following Rapa treatment in older individuals