Investigating the dynamics of surface-immobilized DNA nanomachines

Surface-immobilization of molecules can have a profound influence on their structure, function and dynamics. Toehold-mediated strand displacement is often used in solution to drive synthetic nanomachines made from DNA, but the effects of surface-immobilization on the mechanism and kinetics of this reaction have not yet been fully elucidated. Here we show that the kinetics of strand displacement in surface-immobilized nanomachines are significantly different to those of the solution phase reaction, and we attribute this to the effects of intermolecular interactions within the DNA layer. We demonstrate that the dynamics of strand displacement can be manipulated by changing strand length, concentration and G/C content. By inserting mismatched bases it is also possible to tune the rates of the constituent displacement processes (toehold-binding and branch migration) independently, and information can be encoded in the time-dependence of the overall reaction. Our findings will facilitate the rational design of surface-immobilized dynamic DNA nanomachines, including computing devices and track-based motors

mRNA detektointi käyttämällä DNA hybridisaatioketjureaktiota nanopartikkelien pinnalla

Hybridization chain reaction (HCR) is a powerful tool for the detection of RNA with excellent sensitivity. However, the speed and sensitivity of HCR is usually limited due to its reliance on diffusion kinetics. Constraining the chain reaction in close space allows faster detection speeds and higher sensitivity than non-constrained ones because no diffusion process is needed. The aim of this thesis is to improve the RNA detection speed and sensitivity using HCR. To achieve this, two DNA HCR hairpin monomers were used. After confirming the successful HCR in solution, the HCR monomers were constrained on nanoparticles. The attachment of the DNA hairpins on the surface of the nanoparticles were studied via UV/Vis spectroscopy and verified with transmission electron microscopy. The HCR on top of nanoparticles was studied by measuring time-dependent fluorescence intensity. Successful HCR in solution was verified with polyacrylamide gel electrophoresis and fluorescence measurement and successful attachment of hairpin DNA on top of both gold and silicon nanoparticles was demonstrated. However, the DNA hairpins were partially opened after attachment, which induced the HCR in the absence of the initiator strand. This was verified with polyacrylamide gel electrophoresis. Nevertheless, HCR was proven to be feasible on nanoparticles’ surface and the design needs to be optimized before the method is viable for RNA detection in vivo.Hybridisaatioketjureaktio on tehokas tapa detektoida RNA:ta erinomaisella herkkyydellä. Sen detektionopeus ja -herkkyys on kuitenkin yleensä rajallinen, koska se on riippuvainen diffuusiosta. Ketjureaktion rajoittaminen pieneen tilaan johtaa nopeampiin detektioaikoihin ja tarkempaan herkkyyteen kuin rajoittamattomissa ketjureaktioissa, koska diffuusioilmiötä ei tarvita. Tämän diplomityön tavoite on parantaa hybridisaatioketjureaktion detektionopeutta ja -herkkyyttä. Tavoitteena on yksinkertaistaa synteesiprosessia ja samalla voimistaa fluoresenssisignaalia. Tämän toteuttamiseksi käytettiin kahta DNA hiuspinnimonomeeria. Kun onnistunut hybridisaatioketjureaktio oli todennettu liuoksessa, DNA monomeerit kiinnitettiin nanopartikkeleihin. Hiuspinni-DNA:n kiinnittymistä tutkittiin ultraviolettivalon ja näkyvän valon spektroskopialla ja kiinnitys varmistettiin läpäisyelektronimikroskopialla. Hybridisaatioketjureaktiota nanopartikkelien pinnalla tutkittiin mittaamalla fluoresenssin intensiteetin muutosta. Onnistunut hybridisaatioketjureaktio liuoksessa varmennettiin polyakryyliamidi geelielektroforeesin ja fluoresenssimittauksen avulla. Myös DNA:n onnistunut kiinnittyminen sekä pii- että kultananopartikkelien pinnalle todennettiin. DNA hiuspinnit olivat kuitenkin osittain auenneet kiinnittymisen jälkeen, mikä aiheutti ketjureaktion alkamisen ilman initiaatiotekijää. Aukeaminen varmennettiin polyakryyliamidi geelielektroforeesilla. Tästä huolimatta hybridisaatioketjureaktion todistettiin olevan mahdollinen nanopartikkelien pinnalla. Jotta menetelmästä tulisi käyttökelpoinen RNA:n detektointiin elävissä organismeissa, on menetelmää vielä optimoitava

A DNA-Based Archival Storage System

Abstract Demand for data storage is growing exponentially, but the capacity of existing storage media is not keeping up. Using DNA to archive data is an attractive possibility because it is extremely dense, with a raw limit of 1 exabyte/mm 3 (10 9 GB/mm 3 ), and long-lasting, with observed half-life of over 500 years. This paper presents an architecture for a DNA-based archival storage system. It is structured as a key-value store, and leverages common biochemical techniques to provide random access. We also propose a new encoding scheme that offers controllable redundancy, trading off reliability for density. We demonstrate feasibility, random access, and robustness of the proposed encoding with wet lab experiments involving 151 kB of synthesized DNA and a 42 kB randomaccess subset, and simulation experiments of larger sets calibrated to the wet lab experiments. Finally, we highlight trends in biotechnology that indicate the impending practicality of DNA storage for much larger datasets

Compartmentalization of DNA-Based Molecular Computing Elements Using Lipid Bilayers

This dissertation will present a progression from the detection of double-stranded DNA using a combination of toehold-mediated strand displacement and DNAzyme reactions in dilute saline solutions, to the generation of separate compartments to allow standardization of DNA computing elements, by protecting from complementary strands. In well-mixed solutions complementary regions cause spurious interactions. Importantly, these compartments also provide protection from nucleases. Along the way we will also explore the use of silica microsphere supported lipid bilayers to run compartmentalized DNA reactions on a fluid surface and the design of a molecule capable of DNA-based transmembrane signal transduction

Aplicación de controles de seguridad informática para dispositivos de almacenamiento digital basados en secuencias de ADN

La presente investigación elaboró un modelo común de almacenamiento de información digital basado en secuencias de ADN sintético, a partir de ocho (8) modelos existentes, tomados como muestra, para ello se identificaron treinta y uno (31) actividades comunes, que presentaron dichos modelos, y que permiten cumplir con las dos funcionalidades principales requeridas: la de almacenar información digital en secuencias de ADN y la de extraer la misma información digital de dichas secuencias de ADN. Acto seguido este modelo fue sometido a una evaluación de riesgos de seguridad, aplicando la metodología de la ISO/IEC 27005:2018, dando como resultado la identificación de cuarenta y cinco (45) riesgos de seguridad de la información, las cuales recibieron tratamiento mediante la aplicación de ocho (8) controles de seguridad seleccionados de la ISO/IEC 27002:2013. Al aplicar estos controles de seguridad en el modelo común se agregaron dieciséis (16) actividades de seguridad que permitió garantizar la confidencialidad, disponibilidad e integridad de la información digital, pasando de treinta y un (31) actividades a cuarenta y siete (47) actividades para el modelo seguro de almacenamiento de información digital en moléculas de ADN sintético.The present investigation elaborated a common model of digital information storage based on synthetic DNA sequences, based on eight (8) existing models, taken as a sample, for which thirty-one (31) common activities were identified, which presented these models, and that allow to fulfill the two main required functionalities: that of storing digital information in DNA sequences and that of extracting the same digital information from said DNA sequences. Then this model was subjected to an evaluation of security risks, applying the methodology of ISO / IEC 27005: 2018, resulting in the identification of forty-five (45) information security risks, which were treated by the application of eight (8) selected security controls from ISO / IEC 27002: 2013. By applying these security controls in the common model, sixteen (16) security activities were added that allowed to guarantee the confidentiality, availability and integrity of digital information, going from thirty-one (31) activities to forty-seven (47) activities for the secure model of digital information storage in synthetic DNA molecules.Trabajo de investigaciónCampus Lima Centr