Light-Up Split Broccoli Aptamer as a Versatile Tool for RNA Assembly Monitoring in Cell-Free TX-TL Systems, Hybrid RNA/DNA Origami Tagging and DNA Biosensing

Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a ‘bio-orthogonal’ hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets

자가 조립 와이어프레임 DNA 구조체를 통한 설계 가능한 나노 수준 종이접기 기술 개발

학위논문(석사) -- 서울대학교대학원 : 공과대학 기계공학부, 2022.2. 김도년.The origami mechanism has provided effective solutions to many engineering problems related to structural reconfiguration through its transformable properties. However, the transfer of the origami-based reconfiguration mechanism from macroscale into nanoscale engineering remains a challenge due to the difficulties in effectively implementing high-precision structural design and programming various folding lines (crease patterns) of nanostructures. Here, we developed a nucleic-based crease patterning method on a planar sheet of DNA wireframe nanostructure (DNA wireframe paper) by harnessing the paper folding mechanism and implemented eight reconfigurable folding types of DNA wireframe papers using toehold-mediated strand displacement. The folding yield is optimized above 90% by increasing the binding probability and relieving the structural rigidity of the crease. Based on its high yield secured, folding properties such as orthogonal folding, repeatable folding and unfolding, and folding-based signal control were designed and demonstrated through atomic force microscopy and fluorescence measurements. Furthermore, environmental stimuli-responsive folding according to pH value and UV illumination time was designed and successfully controlled. Moreover, we adopt a hierarchical assembly strategy to program more complex crease patterns and finally achieved 10 types of the intended folding of larger-scale DNA papers polymerized in a quadruple area. With high yield, various programmability, and large scalability, we expect our origami-based structural reconfiguration methods for DNA assemblies to contribute to the advancement of folding-based engineering applications in nanoscale.종이접기 메커니즘은 변형 가능한 특성을 통해 구조 재구성과 관련된 많은 공학 문제들에 대한 효과적인 해결 방안들을 제공해왔습니다. 하지만, 종이접기 기반 구조 재구성 방식을 거시규모에서 나노규모 공학으로 이전하는 것은 정밀한 나노구조체 설계 및 다양한 접힘 (주름 패턴) 설계에 대한 어려움으로 인해 여전히 난제로 남아 있습니다. 본 논문에서는 평면 시트 형태의 DNA 와이어프레임 구조체(DNA 와이어프레임 종이) 위에 종이접기 방식을 접목하여 핵산 기반 주름 설계 방법을 개발하였고 발판 매개 가닥 변위 반응을 통해 DNA 와이어프레임 종이의 8가지의 재구성 가능한 접힘을 구현하였습니다. 접힘 수율은 결합 확률 증대 및 주름의 구조적 강성 완화를 통해 90% 이상으로 최적화 되었습니다. 확보한 높은 수율을 바탕으로 직교 접힘, 반복 접힘과 펼침, 접힘 기반 신호 제어 등 여러가지 접힘 특성들을 설계하였고 원자력 현미경 및 형광 측정을 통해 이를 입증하였습니다. 또한 pH 값 및 자외선 조사에 따른 환경 자극 반응 접힘을 설계해 이를 성공적으로 제어하였습니다. 나아가, 계층적 조립 전략을 채택하여 보다 복잡한 주름 패턴들을 설계하였고, 최종적으로 4배 면적으로 중합된 더 큰 규모의 DNA 종이의 10가지 의도한 접힘을 구현하였습니다. 높은 수율, 다양한 접힘 설계 가능성, 그리고 큰 규모 확장성을 가진 본 연구의 DNA 구조체에 대한 종이접기 기반 구조 재구성 방법이 나노규모에서의 접힘 방식 기반 공학적 응용 분야의 발전에 기여할 것을 기대합니다.Abstract 1 Table of contents 2 List of tables 3 List of notes 3 List of figures 4 Chapter 1. Introduction 6 Chapter 2. Results 8 2.1. Design principle 8 2.2. Yield optimization 33 2.3. Folding properties 49 2.4. Environmental folding control 64 2.5. Larger-scale folding 74 Chapter 3. Conclusion 87 Chapter 4. Materials and methods 88 Appendix 93 Bibliography 101 국 문 초 록 104 Acknowledgments 105석

Computational Design and Study of Structural and Dynamic Nucleic Acid Systems

abstract: DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control. This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.Dissertation/ThesisDoctoral Dissertation Chemistry 201

A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology

Nucleic acid tools for detection and characterization of biological systems

Nucleic acids, DNA and RNA, are naturally occurring biopolymers synthetized by cells to store and propagate genetic information. They can be found in eukaryotic cells, bacteria, archaea and viruses and, thanks to the development of synthetic chemistry techniques, they can be synthetized with relative ease on demand in the laboratory. DNA and RNA can form very distinct structures through Watson-Crick base pairing, where nucleobases form hydrogen bonds between the two antiparallel strands of a double helix. The programmability of base pairs can also be used to create pre-defined structures using nucleic acids as building material. One of the implementations of this, the DNA origami technique is using a long ssDNA oligo (scaffold) and hundreds of shorter oligonucleotides (staples) to bridge different regions of the scaffold together and form well defined shapes. DNA nanostructures generated this way can be used, among other things, as carriers of functional molecules to create patterns. In paper I. we present a method to study the spatial tolerance of antibodies by using DNA origami structures to present nanoscale antigen patterns. The DNA nanopatterns were immobilized on a surface plasmon resonance set up and the binding kinetics of different antibodies were measured. We found that the IgG subclasses and isotypes studied, were able to bind bivalently to two antigens separated by distances between 3 to 17 nm, with a distinct preference showed for the 16nm distance. Different spatial tolerance profiles were observed for a monomeric IgM, and IgG antibodies with lower affinities to antigens. In paper II. we use a DNA origami nanostructure to create different patterns of Jag1 ligand for studying the activation mechanism of the Notch signaling pathway. By treating induced pluripotent stem (iPs) cells with various Jag1 nanopatterns we found that bigger clusters of Jag1, induced more activation of the Notch receptors. This effect was further elucidated to occur because of prolonged binding of the ligand-receptor complex, leading to activation of Notch receptors in the absence of intercellular or external forces. In paper III. we introduce a new method to synthesize DNA origami directly on magnetic beads. Our method, tested for a variety of different DNA origami structures, can achieve up to 90% yield compared to a standard folding protocol. Additionally, the same solid support can be used to functionalize the DNA origami in a one-pot-reaction and purify them from the excess of the molecules. In paper IV. we present a protocol for detecting viral RNA in patient samples with Covid19 by circumventing the RNA extraction step, which was a bottleneck in the detection process at the beginning of the pandemic. Samples were inactivated by heat and the RT-PCR was performed directly (hid-RT-PCR). By comparing our results with the standard diagnostic method on 597 clinical samples we concluded that hid-RT-PCR is a reliable simplified and cost-efficient method that could increase diagnostic availability and subsequent decrease in spread of the virus

Custom-tailored DNA origami mechanics for cellular applications

DNA molecules have been used as the building block for the self-assembly of artificial nanostructures. In particular, the DNA origami method has made the design of DNA nanostructures more robust and approachable. Different design approaches have been created and DNA origami has been used in a variety of fields, from plasmonic, to drug delivery, to biology and biophysics. In recent years, DNA nanotechnology has shown very promising uses in studying forces in biological contexts, both by measuring them and applying them. Mechanosensitive systems in biology are widespread and the study of their complex regulation is increasing in importance, and DNA origami has recently been used as a tool to study them. In paper I, we implement an unsupervised software to simulate wireframe DNA origami structures and evaluate their rigidity. After this evaluation, the software produces mutant structures and then the process is started again, iteratively. In this way the software creates an in-silico evolution towards more rigid wireframe DNA origami. The structures are modified following one of two schemes. In the first one, the individual edges are evaluated and then modified by adding or removing individual bases; in the second scheme, the structures have internal supports, and the software can modify the position of these internal supports to create mutants. We show that these two schemes have different results on the rigidity of the structures, with the internal supports-based scheme increasing the rigidity of structures to up to 50%, after several iterations. In paper II, we compare the mechanical characteristics of a lattice-based DNA origami nanostructure and a wireframe DNA origami nanostructure, exploring how the differences between the two affect their interaction with cancer cells. The wireframe structure showed a higher local flexibility when compared to the lattice-based structure. These physical differences play an important role in the interaction between DNA nanostructures and human cancer cells, in particular thanks to the differences in interaction with scavenger receptors. We show that wireframe origamis are more likely to stay on the cell membrane, while the lattice-based origami are more likely to be internalized. This is also reflected in a deeper penetration of the wireframe structures into cell spheroid tissue models. With these observations, we show that the design method should be considered when applying DNA origami for biological applications. In paper III, we aim to expand the design space of wireframe DNA origami, by designing structures with four-helix bundles (4HB) as edges. This is possible thanks to the addition of additional helices to the edge of the wireframe structures, to create 4HB on a square lattice: this results in increased rigidity of the edges. We developed the software for the design of the new type of structures and then we successfully folded a library of five structures, investigating the rigidity of the new type of structures. In addition, we designed a new type of hybrid structures, presenting more rigid 4HB edges and less rigid single helix edges. We think that the development of new ways of designing DNA origami structures can pave the way for the design of nanostructures more suited for specific applications. In paper IV, we design a DNA origami nanoactuator with the aim of pulling on molecular targets. DNA origami is a promising technology in this field because of its high throughput and the relative simplicity when compared with other force spectroscopy techniques. We designed a barrel-like structure with an internal block connected to ssDNA or dsDNA strands, depending on the activation mode of the mechanism. We estimated that the structure can create forces of up to 40 pN, and coarse-grained molecular dynamics simulations in oxDNA and Förster resonance energy transfer experiments confirm the successful activation of the structure. We also demonstrated that the structure, modified with Cy5, cholesterol, and anti-CD3 aptamer, can interact with T cells. We think that DNA origami can become an important tool in the study of mechanosensitive cellular receptors