DNA sequencing via nanopore translocation was a pipedream two decades ago. Today, biotech companies are releasing commercial devices. Yet many challenges still hover around the simple concept of threading a long DNA molecule through a small nanoscopic pore with the aim of extracting the DNA’s sequence along the process. In this thesis I use computer simulations to create what are in essence virtual pro- totypes for testing design ideas for the improvement of nanopore translocation devices. These ideas are based on the general concept of modifying the average shape of the initial DNA conformations. This is done, for example, by introducing new geometrical features to the nanopore’s surrounding or by the means of some external force. The goal of these simulations is not just to test design improvements, but also to systematically deconstruct the physical mechanisms involved in the translocation process. The roles of pore friction, initial polymer conformations, monomer crowding on the trans- side of the membrane, Brownian fluctuations, and polymer rigidity can, with careful consideration, be essentially muted at will. Computer simulations in this sense play the role of a sandbox in which the physics can be tinkered with, in order to assess and evaluate the magnitude of certain approximations found in theoretical modelling of translocation. This enables me to construct theoretical models that contain the necessary features pertaining to the different designs tested by simulations. The work presented here is thus constituted of both Langevin Dynamics simulations and adaptations of the Tension-Propagation theory of polymer translocation when the polymer is subject to the various test conditions.

Faithful chromosome segregation is essential for all living organisms. Bacterial chromosome segregation utilizes highly conserved directional SpoIIIE/FtsK translocases to move large DNA molecules between spatially separated compartments. These translocases employ an accessory DNA-interacting domain (gamma) that dictates the direction of DNA transport by recognizing specific DNA sequences. To date it remains unclear how these translocases use DNA sequence information as a trigger to expend chemical energy (ATP turnover) and thereby power mechanical work (DNA movement). In this thesis, I undertook a mechanistic study of directional DNA movement by SpoIIIE from the Gram-positive model bacterium Bacillus subtilis. Specifically, I was interested in understanding the information transfer within the protein from sequence recognition, to ATP turnover, and ultimately to chromosome translocation. How do DNA sequences trigger directional chromosome movement?

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Nanopore sensing is a versatile technique that employs very small openings, known as nanopores, to study biomolecules. The use of nanopores on solid-state membranes has gained attention due to its potential for low-cost and high-throughput sensing of single molecules in liquids. Controlled dielectric breakdown (CBD) is a method for fabricating nanopores in a suspended membrane using computer control, and can be performed in liquid, making it a more practical alternative to traditional techniques that require specialized equipment and high vacuum. Multilevel Pulse-Voltage Injection (MPVI) is a variant of CBD that allows for better control over the size and shape of the nanopore being fabricated. The main focus of this research is to develop electrical techniques for fabricating sub-10 nm solid-state nanopores in silicon nitride and graphene membranes, and to study the characteristics of the resulting nanopores. Two different MPVI schemes were implemented for fabricating nanopores in silicon nitride. The MPVI technique for Scheme 1 sets two thresholds to check if a pore is formed or not. Scheme 2 was developed by adding a threshold in order to avoid extra pore enlargement. For nanopores on a silicon nitride membrane with a 23 nm deep hole, the ratio of sub-20 nm pores improved from 20 % (Scheme 1) to around 90 % (Scheme 2). Additionally, the ratio of sub-10 nm nanopores via Scheme 2 was around 70 %. For nanopores on a silicon nitride membrane damaged by a single femtosecond laser pulse, 50 % of the fabricated nanopores via Scheme 2 were sub-10 nm. For bi-layer graphene membranes, the electrochemical reaction (ECR) technique was used to fabricate nanopores, resulting in three nanopores with diameters of 6.4, 5.9, and 1.2 nm. The nanopores on all types of membranes were enlarged using MPVI of Scheme 1, resulting in a successful increase in pore size by 0.1 to 1 nm. Finally, DNA translocation experiments were conducted to verify the suitability of the fabricated nanopores. DNA translocation events were observed using fabricated nanopores on two types of silicon nitride membranes. They are not observed for the graphene nanopore. / Nanopor-avkänning är en mångsidig teknik som använder mycket små öppningar, så kallade nanoporer, för att studera biomolekyler. Användningen av nanoporer på fasta membran har fått uppmärksamhet tack vare dess potential för detektering av enstaka molekyler i vätskor, till lågt pris och med kort genomloppstid. Kontrollerad dielektrisk nedbrytning (CBD) är en metod för att tillverka nanoporer i ett suspenderat membran med hjälp av datorstyrning som kan utföras i vätska, vilket gör den till ett mer praktiskt alternativ till traditionella tekniker som kräver specialiserad utrustning och högt vakuum. Multilevel Pulse-Voltage Injection (MPVI) är en variant av CBD som möjliggör bättre kontroll över storleken och formen på nanoporen som tillverkas. Huvudfokus för denna forskning är att utveckla elektriska tekniker för att tillverka sub-10 nm fasta nanoporer i kiselnitrid och grafenmembran, och att studera egenskaperna hos de resulterande nanoporerna. Två olika MPVI-metoder implementerades för tillverkning av nanoporer i kiselnitrid. MPVI-tekniken i Metod 1 sätter två tröskelvärden för att: kontrollera om en por bildas eller inte, samt för att kontrollera porstorleken. Metod 2 utvecklades genom att lägga till ytterligare ett tröskelvärde för att undvika extra porförstoring. För nanoporer på ett kiselnitridmembran med ett 23 nm djupt hål förbättrades förhållandet mellan porer under 20 nm från 20 % (Metod 1) till cirka 90 % (Metod 2). Dessutom var förhållandet mellan nanoporer under 10 nm med Metod 2 cirka 70 %. För nanoporer på ett kiselnitridmembran som skadats av en femtosekundlaserpuls, även om en nanopor med en diameter under 5 nm inte tillverkades, var 50 % av de tillverkade nanoporerna via Metod 2 under 10 nm. För tvåskiktsgrafenmembran användes den elektrokemiska reaktionstekniken (ECR) för att tillverka nanoporer, vilket resulterade i tre nanoporer med diametrar på 6,4; 5,9 och 1,2 nm. Nanoporerna på alla typer av membran förstorades med MPVI i Metod 1, vilket resulterade i en framgångsrik förstoring av porstorleken med 0,1 till 1 nm. Slutligen genomfördes experiment med DNA-translokation för att verifiera lämpligheten av de tillverkade nanoporerna. DNA-translokationshändelser observerades med hjälp av tillverkade nanoporer på två typer av kiselnitridmembran. De observeras inte för grafen-nanoporen.

The EcoR124INT restriction-modification (R-M) system contains the genes HsdS3, HsdM and HsdR. S3 encodes the N-terminal domain of the wild-type S subunit and has been shown to dimerise in solution (Smith et al., 1998). Following purification of the subunits of the EcoR124INT R-M system, complexes of the methyltransferase S3/M and restriction endonuclease S3/M/R were formed and shown to have activity in vitro, methylating and hydrolysing a symmetrical DNA recognition sequence, respectively. The DNA mimic OCR (overcome classical restriction) protein inhibited the methyltransferase activity in vitro, with maximum inhibition at a 1: 2 molar ratio of (S3/M)2 to an ocr dimer. Dynamic light scattering (DLS), sedimentation equilibrium (SE) and sedimentation velocity (SV) experiments showed S3 to exist as a dimer and S11 (the central conserved domain of S) to exist as a tetramer in solution. M was found to be dimeric in solution, whilst the R protein was monomeric. A complex of S3/M was found to have a stoichiometry (S3/M)2 and a complex of S3/M/R had a stoichiometry of S3/M/R1, even when a 2: 1 molar ratio of R to S3/M, was added. Small angle neutron scattering (SANS) experiments provided values for the radius of gyration (Rg), which for S3 was comparable to that calculated for the recently published crystal structure of the S subunit from Methanococcus jannaschii (Kim et al., 2005). These experiments also showed a decrease in the Dmax in the presence of the 30 bp DNA recognition sequence from 200A to 140A, suggesting a similar conformational change in the positioning of the subunits as has been detected for the wild-type M. EcoR124I and a related type 1 1/2 system AhdI. This change following DNA binding was also observed by SV experiments. Furthermore ab initio modelling from the SANS data has provided a low-resolution structure for the EcoR124INT MTase and its complex with DNA.

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown. Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we build a structural model for TerL pentamer assembly. Apo and ATP analog-bound TerL ATPase domain crystal structures show ligand-dependent conformational changes, which we propose power DNA translocation. Together, we assimilate these findings to build models for both motor assembly and DNA translocation. Additionally, with the P76-26 system, we identify the TerS protein as gp83. I find that P74-26 TerS is a nonameric ring that stimulates TerL ATPase activity while inhibiting TerL nuclease activity. Using cryoEM, I solve 3.8 Å and 4.8 Å resolution symmetric and asymmetric reconstructions of the TerS ring. I observe in P74-26 TerS, the conserved C-terminal beta-barrel is absent, and instead the region is flexible or unstructured. Furthermore, the helix-turn-helix motifs of P74-26 TerS are positioned differently than those of known TerS structures, suggesting P74-26 uses an alternative mechanism to recognize DNA.