Multivalent aptamer refers to a technique that joins two or more identical or different types of aptamer monomers together, with or without the presence of structural or other functional elements. As a rapid and easy method for fabricating the multivalent aptamer constructs, rolling circle amplification (RCA) has attracted great attention in recent decades. The incorporation of properly designed structural elements, such as intra-molecular spacers, have been shown to greatly enhance the efficiency of the multivalent aptamer system [1]. The objective of this current study is to systemically investigate the effect of different lengths of poly thymine spacer designs (polyT, from no spacer/NT, 5T, 10T, and 15T) on the capturing performance of RCA-generated multivalent system. To achieve this, we designed four circular probe templates by inserting zero, five, ten, and fifteen adenine bases (polyA). These polyA domains in the circular probe template are complementary to polyT with respective lengths in between the adjacent aptamers on the resultant RCA products (RCAPs). We found that the resultant RCAPs with length shorter than 10T showed a lack or low ability to capture target cells E.Coli O157:H7. When spacer lengths reach or exceed 10T, the capturing performance of the respective multivalent aptamer chain is significantly enhanced. This phenomenon can be explained by larger hydrodynamic sizes and less nonspecific secondary structures observed in RCAP with spacer length no less than 10T. Moreover, we found that there is also a trade-off that the number of polyA bases added into the circular probe template can significantly impair the efficiency of RCA reaction in respective to cyclization yield and amplification rate. The results of this research explain with details how the design of spacer affects RCA reaction efficiency and RCAPs’ capturing performance, which provides ideas in designing an efficient RCA-generated multivalent system.

The advance of biological research promotes the emerging of new methods and solutions to answer the biological questions. This thesis describes several new molecular tools and their applications for the detection of genomic and proteomic information with extremely high sensitivity and specificity or simplify such detection procedures without compromising the performance. In paper I, we described a general method namely super RCA, for highly specific counting of single DNA molecules. Individual products of a range of molecular detection reactions are magnified to Giga-Dalton levels that are easily detected for counting one by one, using methods such as low-magnification microscopy, flow cytometry, or using a mobile phone camera. The sRCA-flow cytometry readout presents extremely high counting precision and the assay’s coefficient of variation can be as low as 0.5%. sRCA-flow cytometry readout can be applied to detect the tumor mutations down to 1/100,000 in the circulating tumor cell-free DNA. In paper II, we applied the super RCA method into the in situ sequencing protocol to enhance the amplified mRNA detection tags for better signal-to-noise ratios. The sRCA products co-localize with primary RCA products generated from the gene specific padlock probes and remain as a single individual object in during the sequencing step. The enhanced sRCA products is 100% brighter than regular RCA products and the detection efficiency at least doubled with preserved specificity using sRCA compared to standard RCA. In paper III, we described a highly specific and efficient molecular switch mechanism namely RCA reporter. The switch will initiate the rolling circle amplification only in the presence of correct target sequences. The RCA reporter mechanism can be applied to recognize single stranded DNA sequences, mRNA sequences and sequences embedded in the RCA products. In paper IV, we established the solid phase Proximity Ligation Assay against the SOX10 protein using poly clonal antibodies. Using this assay, we found elevated SOX10 in serum at high frequency among vitiligo and melanoma patients. While the healthy donors below the threshold.

The development of easily fabricated, highly stable DNA-based microarray and continuous flow concentrating devices is vital for several biomedical and environmental applications. Nucleic acid biosensors can be used for genetic analysis, disease diagnosis, drug discovery, food and water quality control and more, however methods of fabrication are tedious, and the longevity of sensors is compromised by the fragility of the sensing component. In this report, the fabrication and characterization of two biosensing modalities – microarrays and microgels – composed of Rolling Circle Amplification (RCA) product in poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels are investigated. RCA product microarrays were developed by the sequential printing of aldehyde and hydrazide functionalized POEGMA precursors on nitrocellulose paper, exploiting rapid gelling via hydrazone crosslinking to generate thin film hydrogel sensing arrays. POEGMA/RCA product microgels for affinity column applications were synthesized using an inverse emulsion polymerization technique. Inkjet printing evenly deposited RCA product in all wells, with POEGMA effectively stabilizing DNA on the cellulose substrate. Hybridization of complementary probe to the encapsulated RCA product was optimized, yielding a signal to noise ratio of ~4 for a large range of probe concentrations. Microgels were successfully synthesized in the size range of 10-60 μm diameter, and a linear model that can accurately predict size based on initiator and emulsifier concentration was developed. The encapsulation efficiency of RCA product in different sized microgels was explored, with larger microgels entrapping more product and the highest encapsulation efficiency calculated at 56%. These results demonstrate that POEGMA hydrogels can be utilized to encapsulate and stabilize RCA product in two distinct structures, providing a basis for the development of easily fabricated biosensors for more specific applications. / Thesis / Master of Applied Science (MASc)

The technique of DNA origami utilizes the specific and limited bonding properties of DNA to fold single stranded DNA sequences of various lengths to form a predesigned structure. One longer sequence is used as a scaffold and numerous shorter sequences called staples, which are all complementary to the scaffold sequence, are used to fold the scaffold into intricate shapes. The most commonly used scaffold is derived by extracting the genome of the M13 phage and the staples are usually chemically synthesized oligonucleotides. Longer single stranded sequences are difficult to synthesize with high specificity, which limits the choices of scaffold sequences available. In this project two main methods of single stranded amplification, Rolling Circle Amplification (RCA) and the usage of helper phages, were explored with the goal to produce both a 378 nt scaffold and staple sequences needed for folding a DNA origami structure. To facilitate imaging by Transmission Electron Microscopy (TEM) of this small structure, the DNA origami structure was created to form a polymer structure. Production of the scaffold sequence in high yield was unsuccessful and no well-defined polymers were found in the folded samples, though a few results showed promise for further studies and optimizations. Due to time constraints of this project, only production of the scaffold sequence was tested. Unfortunately the scaffold produced by the helper phages was of the complementary strand to that used to design the DNA origami structure, and could therefore not be used for folding. The correct strand was produced by the RCA where the yield was too low when using Phi29 DNA polymerase for proper folding to take place, though small scale RCA by Bst DNA polymerase on the other hand showed promising results. These results indicate that the scaffold production may not be far off but still more experience in producing intermediate size oligonucleotides may be necessary before succeeding in high yield production of this 378 nt long sequence. The promise given by this production is to enable high yield, high purity, low cost and also an easily scalable process set-up. This would be an important step in future DNA nanotechnology research when moving from small scale laboratory research to large scale applications such as targeted drug delivery systems.

Treatment success often depends on the availability of accurate and reliable diagnostic assays to guide clinical practitioners in their treatment choices. An optimal test must excel in specificity and sensitivity, and depending on the application area time, low-cost and simplicity are equally important. For instance, time is essential in infectious diagnostics but this is less important in non-invasive prenatal testing (NIPT). In NIPT, specificity and sensitivity are the most important parameters. In this thesis I describe the development of four different methods, all based on padlock probes and rolling circle amplification, intended for molecular diagnostics. Application areas range from infectious disease diagnostics to NIPT and oncology. The methods described have in common that they overcome certain limitations of currently available assays. This thesis includes two new assays targeting infectious agents: one assay specifically detecting a highly variable double stranded RNA virus and the second assay demonstrating a new format of antibiotic susceptibility testing, which is rapid and generally applicable to different pathogens. Furthermore, I describe the development of a method that uses methylation markers to enrich fetal DNA, accurately quantify chromosome ratios and thus, detecting trisomy 21 and 18. The fourth method described in this thesis uses gap-fill ligation of padlock probes to detect diagnostic relevant point mutations with high specificity in situ. The assays presented have the potential, after automation and successful validation and verification studies, to be implemented into clinical practice. Furthermore, these assays demonstrate the wide applicability of padlock probes which, due to their properties in regard to specificity and multiplexity, are useful tools for nucleic acid detection in vitro as well as in situ. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

RNA-cleaving DNAzymes (RCDs) are a class of functional nucleic acids that can bind various targets ranging in size from small molecules to large proteins, which results in activation of cleavage activity. The activation of RCDs results in the cleavage of a ribonucleotide site in an otherwise all-DNA substrate, leading to two cleavage fragments. In this work, a previously identified DNAzyme that binds to a protein biomarker endogenous to Helicobacter pylori (J99) crude extracellular matrix was evaluated for coupling to an isothermal amplification method termed rolling circle amplification (RCA) as a way to improve the originally reported detection limit. Three RCD constructs were designed with the goal of generating a cleavage fragment that could act as a primer to initiate RCA. The first method used the original HP DNAzyme, which liberated a short cleavage fragment that could be used as a primer. However, the primer fragment was rapidly digested by the bacterial matrix, preventing RCA. A second method evaluated use of a circularized substrate and separate RCD to generate a primer, however this system was not capable of generating a cleavage fragment. A final method redesigned the original RCD to move the substrate region from the 3’ to the 5’ end of the RCD, causing the longer RCD-containing fragment to be the primer for RCA. In this case, target-triggered cleavage was observed and the resulting primer was sufficiently resistant to digestion to allow its use as a primer for RCA. Preliminary characterization of the rearranged RCD showed that it retained selectivity similar to the original RCD, but that the cleavage rate was slower. In addition, the RCA based reaction, while successful, did not produce improved detection sensitivity relative to unamplified assays. Methods to further improve RCA performance are discussed for future work. / Thesis / Master of Science (MSc)

<p>Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges.</p><p>The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.</p>

This thesis describes a new approach to biomolecular analysis, called the volume-amplified magnetic nanobead detection assay (VAM-DNA). It is a sensitive, specific magnetic bioassay that offers a potential platform for the development of low-cost, easy-to-use diagnostic devices. The VAM-NDA consists of three basic steps: biomolecular target recognition, enzymatic amplification of the probe-target complex using the rolling circle amplification (RCA) technique, and addition of target complementary probe-tagged magnetic nanobeads which exhibit Brownian relaxation behavior. Target detection is demonstrated by measuring the frequency-dependent complex magnetization of the magnetic beads. The binding of the RCA products (target DNA-sequence coils) to the bead surface causes a dramatic increase in the bead size, corresponding essentially to the size of the DNA coil (typically around one micrometer). This causes a decrease in the Brownian relaxation frequency, since it is inversely proportional to the hydrodynamic size of the beads. The concentration of the DNA coils is monitored by measuring the decrease in amplitude of the Brownian relaxation peaks of free beads. The parameters oligonucleotide surface coverage, bead concentration, bead size and RCA times were investigated in this thesis to characterize features of the assay. It was found that all of these parameters affect the outcome and efficiency of the assay. The possibility of implementing the assay on a portable, highly sensitive AC susceptometer platform was also investigated. The performance of the assay under these circumstances was compared with that using a superconducting quantum interference device (SQUID); the sensitivity of the assay was similar for both platforms. It is concluded that, the VAM-NDA opens up the possibility to perform biomolecular detection in point-of-care and outpatient settings on portable platforms similar to the one tested in this thesis. Finally, the VAM-NDA was used to detect Escherichia coli bacteria and the spores of Bacillus globigii, the non-pathogenic simulant of Bacillus anthracis. A limit of detection of at least 50 bacteria or spores was achieved. This shows that the assay has great potential for sensitive detection of biomolecules in both environmental and biomedical applications.

Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges. The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.

The in situ proximity ligation assay (in situ PLA) is an immunoassay that enables directvisualisation of single protein targets or protein interactions in cell or tissue samples. This project revolves around designing and introducing several novel multiplexable components tobe used in conjunction with Olink Bioscience's Duolink product line. In this report, a novel in silico approach to DNA oligomer interaction design is presented. Using this in silico method, a multiplexed system of DNA oligomers has been designed andevaluated using in situ PLA and fluorescence microscopy.