This thesis demonstrates the application of biosensor technology for molecular biology investigations, utilizing a surface plasmon resonance based optical device for mass sensitive detection of biomolecular interactions at a chipsurface. Oligonucleotide model systems were designed for analysis of the action of DNA manipulating enzymes. DNA ligation, DNA cleavage and DNA synthesis could be quantitatively monitored in real-time. A protocol for DNA minisequencing was also established based on prevention of chain elongation by incorporation of chain-terminators. Determinations of affinities for short oligonucleotides hybridizing to an immobilized target were performed with various sequence content, length, temperature and degree of complementarity. The decrease in affinity for hybridizations involving mismatch situations was found to be strongly dependent on the relative position of the mismatch. Interestingly, also end-mismatches were clearly detectable. The stabilization effect achieved upon co-hybridization of two adjacently annealing short oligonucleotide modules (modular primer effect) was also investigated for different module combinations and hybridization situations. The modular concept of hybridizations was subsequently demonstrated to result in enhanced Capture of single stranded PCR products. The sequence based DNA analysis, first introduced with oligonucleotide modelsystems, was extended to the scanning and screening formutations in PCR amplified DNA from clinically relevant samples. Several different formats were investigated, eitherwith the PCR products immobilized on the chip and oligonucleotides injected or vice versa. Again, mismatch discrimination could be observed for wild type and mutant specific oligonucleotides hybridizing to the targets. The experimental set-up for mutation detection was further developed by the introduction of a subtractive mismatch sensitive hybridization outside the instrument and a subsequent determination of the relative amounts of remain ingoligonucleotides with analytical biosensor monitoring of hybridizations between fully complementary oligonucleotides. In conclusion, the applied technology was found to be a suitable tool for a wide range of molecular biology applications, with emphasis on hybridization analysis and mutation detection. / QC 20100611

Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.

Ductal carcinoma in situ (DCIS); is thought directly to precede invasive breast cancer (IBC). Screening mammography has driven the incidence of this key precursor lesion to >65,000 cases per year. However, little is known about the factors controlling the natural history or risk for recurrence following treatment of a particular patients DCIS. Though the heterogeneity of the disease is well established, no histologic or demographic criteria have been able to stratify DCIS for treatment. We hypothesize that at initial diagnosis there exist biologically distinct subsets of DCIS with associated prognoses that may be recognized by molecular markers. Molecular approaches have been limited by technical design issues related to the types of tissue available for analysis, namely degraded formalin-fixed paraffin embedded (FFPE) specimens and small core biopsy samples. However, new technologies promise to overcome these issues. In the first phase of our investigation, we aimed a) to pilot feasibility studies on the use of FFPE DCIS for molecular analyses including gene expression microarray and b) to pilot feasibility study of selective, high throughput sequencing through the use of "exon capture" on small input material that simulated expected DCIS core biopsy amounts. The results of this work offer specific technical guidelines for the molecular study of DCIS. Moreover, they have enabled the initiation of the second phase of this study, which aims to assess molecular profiles of DCIS recurrence and progression.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 178-195).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Uveal melanoma is the most common primary intraocular malignant tumour in adults. Despite improvements in the diagnosis and treatment of the primary tumour, patients continue to have the same mortality rate as several decades ago, reflecting our poor understanding of the mechanisms behind the formation of metastases in this disease. The purpose of this study was therefore to characterize an animal model of uveal melanoma and use this model to study the transcriptional changes that cells undergo from culture to intraocular tumour, to circulation and finally to the formation of a metastatic nodule. / Using microarrays we identified 314 changes in transcript abundance between the intraocular tumour and metastatic lesions. Principal Components Analysis was used to cluster these transcripts into four distinct groups. A further 61 gene transcripts showed statistically significant changes between re-cultured cells isolated from the model, with the circulating malignant cells representing an intermediate step between cells isolated from intraocular tumours and metastatic lesions. We have produced a detailed analysis of the molecular changes that take place as human uveal melanoma cells evolve from a primary tumour to metastasis in an animal model, including the decrease in expression of specific melanoma markers. These changes were verified using quantitative real time polymerase chain reaction and three different functional assays. / In addition we sought to describe the genetic changes that are present in these cells. Using comparative genomic hybridization arrays we were able to successfully describe the deletions and amplifications that are present in genomic DNA extracted from paraffin embedded sections of the primary tumour. This represents the first time that archival tissue has successfully been used for this sort of analysis in uveal melanoma. We identified several genomic amplifications and deletions including an area of amplification of Wnt2, which is involved in beta-catenin regulation and C-Met, which plays a role in tumour cell homing to the liver in patients. / To the best of our knowledge, this is the first time that a detailed genetic analysis has been carried out on the progression of uveal melanoma from intraocular tumour, to circulation, to the formation of metastases.

The sequencing of the human genome revealed that the number of genes does not explain why humans are different from other organisms like mice and dogs. Instead, it is how genes interact with each other and the environment that separates us from other organisms. This motivates the study of genetic networks and, consequently, my research. My work delves into the roles that simple genetic networks play in a cell and explores the biotechnological aspects of how to uncover such genes and their interactions in experimental models. / Cells must respond to the extracellular environment to contract, migrate, and live. Cells, however, are subject to stochastic fluctuations in protein concentrations. I investigate how cells make important decisions such as gene transcription based on noisy measurements of the extracellular environment. I propose that genetic networks perform Bayesian inference as a way to consider the probabilistic nature of these measurements and make the best decision. With mathematical models, I show that allosteric repressors and activators can correctly infer the state of the environment despite fluctuating concentrations of molecules. Viewing transcriptional networks as inference modules explains previous experimental data. I also discover that the particular inference problem determines whether repressors or activators are better. / Next, I explore the genetic underpinnings of two canine models of atrial fibrillation: atrial tachypacing and ventricular tachypacing. Using Affymetrix microarrays, I find that the genetic signatures of these two models are significantly different both in magnitude and in class of genes expressed. The ventricular tachypacing model has thousands of transcripts differentially expressed with little overlap between 24 hours and 2 weeks, suggesting independent mechanisms. The atrial tachypacing model demonstrates an adaptation as the number of genes found changed decreases with increasing time to the point that no genes are changed at 6 weeks. I use higher level analysis to find that extracellular matrix components are among the most changed in ventricular tachypacing and that genes like connective tissue growth factor may be responsible. / Finally, I generalize the main problem of microarray analysis into an evaluation problem of choosing between two competing options based on the scores of many independent judges. In this context, I rediscover the voting paradox and compare two different solutions to this problem: the sum rule and the majority rule. I find that the accuracy of a decision depends on the distribution of the judges' scores. Narrow distributions are better solved with a sum rule, while broad distributions prefer a majority rule. This finding motivates a new algorithm for microarray analysis which outperforms popular existing algorithms on a sample data set and the canine data set examined earlier. A cost analysis reveals that the optimal number of judges depends on the ratio of the cost of a wrong decision to the cost of a judge.

Mutacijos, atsiradusios atitinkamuose žmogaus genuose, gali lemti pakitusių baltymų atsiradimą, kurie sukelia įvairias ligas (pvz.: vėžį), klaidingą embriono vystymąsi ar priešlaikinę mirtį. Tokios genetinės ligos gali būti gydomos genų terapijos būdu. Labiausiai vystoma genų terapijos strategija yra paremta homologine rekombinacija, kurios metu DNR seka, naudojama geno taisymui, yra patiekiama in trans. Natūraliai žinduolių ląstelėse homologinė rekombinacija (HR) vyksta žemu rekombinacijos dažniu (10-6). Tačiau yra žinoma, kad dvigrandininio trūkio įvedimas žymiai pagreitina HR (10-1). In vivo eksperimentų atveju dvigrandininio trūkio įvedimas turi būti ypač tikslus, todėl šis metodas reikalauja naujų molekulinių įrankių, kurie būtų itin specifiški ir griežtai kontroliuojami. Šiame darbe mes orientavomės į itin specifiškų ir griežtai kontroliuojamų meganukleazių kūrimą naudojant restrikcijos endonukleazių (REazių)-tripleksą formuojančių oligonukleotidų (TFO) konjugatus. REazių-TFO konjugatuose TFO suteikia specifiškumą prailgintam atpažinimo taikiniui per DNR triplekso susidarymą taip nukreipdamas restrikcijos fermentą prie konkretaus taikinio kur norima įvesti dvigrandininį trūkį. Šiuo tyrimu mes parodėme dvi alternatyvias restrikcijos endonukleazių-TFO konjugatų aktyvumo reguliavimo strategijas, kas leistų šias nukleazes panaudoti in vivo tyrimuose. Tuo tikslu buvo pasirinkti ortodoksiniai restrikcijos fermentai MunI ir Bse634I, kurie mūsų laboratorijoje yra gerai... [toliau žr. visą tekstą] / Simple mutations within the coding region of critical human genes can lead to the formation of abnormal proteins, resulting in various diseases (e.g. cancer), in failure of an embryo to develop, or premature death. Genetic diseases can only be truly cured via restoration of defective gene function and one of the most promising strategies is based on homologous recombination. Naturally homologous recombination occurs with a low frequency (1 in 106 transfected cells), however it is known that DNA double-strand breaks enhance the efficiency of homologous recombination by several orders of magnitude (up to 10,000-fold). Therefore, gene therapy via homologous recombination requires new molecular tools that should be highly specific and rigorously controllable. In this work we have focused on the development of restriction enzyme-triple helix forming oligonucleotide (TFO) conjugates, where TFO provides specificity for the extended recognition site through the triple helix formation and addresses restriction enzyme to a particular target site where it introduces a double stranded break. We provide proof-of-concept demonstrations of two alternative strategies to control the DNA cleavage activity of restriction endonuclease-TFO conjugates, that allows adopt them in in vivo experiments. To this end we used restriction endonucleases MunI and Bse634I, which were structurally and biochemically characterized before in our laboratory. We successfully combined the restriction endonuclease... [to full text]

Simple mutations within the coding region of critical human genes can lead to the formation of abnormal proteins, resulting in various diseases (e.g. cancer), in failure of an embryo to develop, or premature death. Genetic diseases can only be truly cured via restoration of defective gene function and one of the most promising strategies is based on homologous recombination. Naturally homologous recombination occurs with a low frequency (1 in 106 transfected cells), however it is known that DNA double-strand breaks enhance the efficiency of homologous recombination by several orders of magnitude (up to 10,000-fold). Therefore, gene therapy via homologous recombination requires new molecular tools that should be highly specific and rigorously controllable. In this work we have focused on the development of restriction enzyme-triple helix forming oligonucleotide (TFO) conjugates, where TFO provides specificity for the extended recognition site through the triple helix formation and addresses restriction enzyme to a particular target site where it introduces a double stranded break. We provide proof-of-concept demonstrations of two alternative strategies to control the DNA cleavage activity of restriction endonuclease-TFO conjugates, that allows adopt them in in vivo experiments. To this end we used restriction endonucleases MunI and Bse634I, which were structurally and biochemically characterized before in our laboratory. We successfully combined the restriction endonuclease... [to full text] / Mutacijos, atsiradusios atitinkamuose žmogaus genuose, gali lemti pakitusių baltymų atsiradimą, kurie sukelia įvairias ligas (pvz.: vėžį), klaidingą embriono vystymąsi ar priešlaikinę mirtį. Tokios genetinės ligos gali būti gydomos genų terapijos būdu. Labiausiai vystoma genų terapijos strategija yra paremta homologine rekombinacija, kurios metu DNR seka, naudojama geno taisymui, yra patiekiama in trans. Natūraliai žinduolių ląstelėse homologinė rekombinacija (HR) vyksta žemu rekombinacijos dažniu (10-6). Tačiau yra žinoma, kad dvigrandininio trūkio įvedimas žymiai pagreitina HR (10-1). In vivo eksperimentų atveju dvigrandininio trūkio įvedimas turi būti ypač tikslus, todėl šis metodas reikalauja naujų molekulinių įrankių, kurie būtų itin specifiški ir griežtai kontroliuojami. Šiame darbe mes orientavomės į itin specifiškų ir griežtai kontroliuojamų meganukleazių kūrimą naudojant restrikcijos endonukleazių (REazių)-tripleksą formuojančių oligonukleotidų (TFO) konjugatus. REazių-TFO konjugatuose TFO suteikia specifiškumą prailgintam atpažinimo taikiniui per DNR triplekso susidarymą taip nukreipdamas restrikcijos fermentą prie konkretaus taikinio kur norima įvesti dvigrandininį trūkį. Šiuo tyrimu mes parodėme dvi alternatyvias restrikcijos endonukleazių-TFO konjugatų aktyvumo reguliavimo strategijas, kas leistų šias nukleazes panaudoti in vivo tyrimuose. Tuo tikslu buvo pasirinkti ortodoksiniai restrikcijos fermentai MunI ir Bse634I, kurie mūsų laboratorijoje yra gerai... [toliau žr. visą tekstą]

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).