Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases.

The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC₅₀ more than 20 times lower than that of GRN163L.

In this thesis two different assays for real-time RNA-cleaving deoxyribozyme and general nuclease kinetics are presented. Previous publications on nuclease kinetic assays have been riddled with drawbacks of labeling, discontinuity, cost etc. To tackle some of the drawbacks two assays were developed; the first specifically for RNA-cleaving deoxyribozymes to allow real-time kinetic measurements independently of whether the deoxyribozyme has low or high levels of secondary structure and when cleaving a full length messenger RNA (mRNA) substrate; the second assay was developed as a means to measure kinetics of virtually any nuclease by utilizing the single ubiquitous phenomenon in nuclease cleavage, the exposure of a phosphate upon hydrolysis of the phosphate backbone.

In Paper I the assay for RNA-cleaving deoxyribozyme kinetics is presented as a development of a previously published assay. The search for a fluorescent intercalating dye with more preferential properties than ethidium bromide resulted in PicoGreen. This dye allowed the assay to be used for deoxyribozymes with low and high levels of secondary structure as well as using full length mRNA substrates.

Paper II presents the second assay of this thesis, an assay where phosphates exposed by nuclease cleavage are released from their products by phosphatases; the released inorganic phosphates are quantified in real-time by a biosensor. The assay allows for real-time kinetics without the use of labels (i.e. natural enzymes and substrates). Regardless of whether the nuclease was a protein, nucleic acid-based, an exo- or endonuclease, processive or single-target nuclease the assay suited them equally well.

Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

Determining kinetic constants is important in the field of RNA-cleaving deoxyribozymes (DNAzymes). Using todays conventional gel assays for DNAzyme assays is time-consuming and laborious. There have been previous attempts at producing new and improved assays; however these have drawbacks such as incompatibility with structured DNAzymes, enzyme or substrate modifications and increased cost. Here we present a new method for determining single-turnover kinetics of RNA-cleaving DNAzymes in real-time and in a high-throughput fashion. The assay is based on an intercalating fluorescent dye, PicoGreen, with high specificity for double-stranded DNA and heteroduplex DNA-RNA in this case formed between the DNAzyme and the target RNA. The fluorescence decreases as substrate is converted to product and is released from the enzyme. Using a Flexstation II multi-mode plate reader with built in liquid handling we could automate parts of the assay. This assay gives the possibility to determine single-turnover kinetics for up to 48 DNAzymes simultaneously. As the fluorescent probe is extrinsic there is no need for enzyme or substrate modifications, making this method less costly compared to other methods. The main novelty of this assay is the possibility of using full-length mRNA as the DNAzyme target.

At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.

The field of gene therapy is starting to move towards clinical applications but is currently limited by the lack of efficient delivery systems. Cell-penetrating peptides provide a means of cellular delivery for gene therapy applications as well as delivery of traditional drugs. Using cell-penetrating peptides a range of different cargos have been successfully delivered into a number of cell types, in vitro as well as in vivo. In this review we discuss uptake mechanisms of different cell-penetrating peptides, with or without cargo. The transition from in vitro to in vivoapplications and strategies to increase the bioavailability of cell-penetrating peptides are also discussed.

In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect He La cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA.5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.