Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of ±0.7 µm or ±2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension

Mimicking the action of ribonucleases : studies on RNase A and design of PNA based artificial enzymes

A 3’-deoxy-3’-C-methylenephosphonate modified diribonucleotide is highly resistant to degradation by spleen phosphodiesterase and not cleaved at all by snake venom phosphodiesterase. Despite that both the vicinal 2-hydroxy nucleophile and the 5’-oxyanion leaving group are intact, the 3’-methylenephosponate RNA modification is also highly resistant towards the action of RNase A. Several different approaches were explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. Using a post PNA-assembly procedures oligoether attachment to both N-terminal and sidechain amino groups was achieved. Use of a new oligoether functionalized amino acid allows inclusion of oligoether conjugates during on-line machine assisted synthesis, allowing combination of methods for attachment of different oligoethers and co-conjugation of neocuproine cleaver. We have previously shown that PNA-neocuproine conjugates can act as artificial RNA restriction enzymes (PNAzyme). In the present study we have additionally conjugated the PNA with different entities and also constructed systems where the PNA is designed to clamp the target RNA forming a triplex. Some conjugations are detrimental for the activity while most are silent which means that conjugation can be done to alter physical properties without losing activity. Conjugation with a single oligoether close to the neocuproine does enhance the rate almost two folds compared to the system without the oligoether. The systems designed to clamp the RNA target by forming a triplex are effective if the clamping part is not too long. Changing the direction of a closing base pair, from a GC to a CG pair, enhances the rate of cleavage with a clamping PNAzyme and without compromising the selectivity, leading to the so far most efficient artificial nuclease reported. Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific metal-free RNA cleavers. Here we investigate conjugates with peptide nucleic acids (PNA). In a first study we show that RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates. In a second study we show that tris(2-aminobenzimidazole) based artificial nucleases cleave RNA substrates, which form a bulge upon binding to the PNA, with turnover of substrate and a cleavage rate that is also dependent on the bulge sequence. Two methods of analysis for the kinetics, based on IE-HPLC separation of oligonucleotide fragments and analysis of Cy5-labelled oligonucleotide fragments by denaturating PAGE on a DNA sequencer respectively are also compared. To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have looked at BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs

Target Alkylation of Single and Double Strand DNA by Peptide Nucleic Acids

Quinone methides (QMs) generated in vivo can alkylate DNA and function as anti-cancer drugs. Delivery of QMs to target DNA is necessary to reduce the side effects caused by indiscriminate reaction. Previous, DNA was conjugated with a QM and was successfully used to deliver this QM to complementary DNA sequences. Peptide nucleic acids (PNAs) conjugates of QM are now being developed for in vivo application since PNA binds to its complementary DNA or RNA and PNA resists degradation by nucleases and proteases. The PNA1-QMP1 conjugate is capable of alkylating more than 60% of a complementary ssDNA when added at nearly stoichiometric quantities. No alkylation was observed if non-complementary DNA was treated with the conjugate. PNA1-QMP1 can alkylate a non-complementary DNA only when both the PNA and DNA target bind to a template strand. When no target sequences were present in solution, QM can react with nucleophiles from PNA1 and generate PNA1-QM1 self adduct. ssDNA can be alkylated by PNA1-QM1 self adduct with a 40% yield. The self adduct can survive after an incubation for 7 days in aqueous solution and preserve half of its original ability to alkylate complementary DNA. The reversibility and stability of the self adduct suggest that it can be used in cells. ssRNA can also be recognized and modified by PNA conjugates with a similar yield as earlier demonstrated with ssDNA. A PNA1-QM1 self adduct may also function as a telomerase inhibitor by alkylating RNA within telomerase. Polypyrimidine PNAs were prepared to bind to the major groove of duplex DNA selectively and expand the potential targets from single to double strand DNA. A cytosine-rich PNA recognized dsDNA and delivered an electron-rich QMP2 to its target sequences. The polypurine strand within a target dsDNA was alkylated at 37°C with a yield of 26%. PAN-QMP2 also showed strong selectivity toward its fully matched dsDNA over one base mismatch in the triplex recognition site. Successful delivery of a QMP to target single and double strand DNA by PNAs confirms that the use of PNA in vivo to target pre-selected sequences is feasible

Development of PNA-based systems for food safety and molecular biology

Il presente lavoro di tesi è stato incentrato sullo sviluppo di metodi per applicazioni riguardanti la sicurezza alimentare e la biologia molecolare. L’analisi di norovirus è stata studiata mediante metodi basati sull’utilizzo di Acidi Peptido Nucleici (PNA): i) un PNA-microarray è stato sviluppato per l’analisi e la determinazione del genogruppo di norovirus; ii) sonde a PNA coniugate con arancio di tiazolo (TO) sono state sviluppate ed utilizzate per l’analisi di norovirus mediante un metodo isotermo di amplificazione, analizzando gli amplificati costituiti da RNA; iii) un nuovo approccio per la concentrazione di norovirus dall’acqua e sucessiva analisi mediante real time RT-PCR è stato investigato. Più in generale, sonde a PNA coniugate con pirene sono state sviluppate per l’analisi di sequenze specifiche e per la determinazione di ‘polimorfismi a singolo nucleotide’ (SNPs). In particolare, un sistema basato su una coppia di sonde è stato utilizzato per l’analisi del polimorfismo PTPN22 C1858T, implicato in svariate malattie autoimmuni incluso il diabete di tipo 1.The work has been focused on the development of molecular methods for food safety and molecular biology applications. Norovirus detection has been investigated by Peptide Nucleic Acid (PNA)-based methods: i) a PNA-microarray has been developed and used for the detection and genogroup differentiation of norovirus; ii) thiazole orange (TO) conjugated PNA-probes have been developed and employed for norovirus analysis using an isothermal-based amplification method, targeting RNA; iii) a new approach for the concentration of norovirus from water followed by real time RT-PCR analysis has been investigated. In a more general approach, pyrene-conjugated PNA probes have been developed for the analysis of specific sequences and single nucleotide polymorphism (SNP) detection. In particular, a dual-probe system has been developed for the detection of the PTPN22 C1858T polymorphism implicated in several human autoimmune diseases including type 1 diabetes