Metal-Ion-Binding Oligonucleotides: High-Affinity Probes for Nucleic Acid Sequences

Small non-coding RNAs have numerous biological functions in cell and are divided into different classes such as: microRNA, snoRNA, snRNA and siRNA. MicroRNA (miRNA) is the most studied non-coding RNA to date and is found in plants, animals and some viruses. miRNA with short sequences is involved in suppressing translation of target genes by binding to their mRNA post-transcriptionally and silencing it. Their function besides silencing of the viral gene, can be oncogenic and therefore the cause of cancer. Hence, their roles are highlighted in human diseases, which increases the interest in using them as biomarkers and drug targets. One of the major problems to overcome is recognition of miRNA. Owing to a stable hairpin structure, chain invasion by conventional Watson-Crick base-pairing is difficult. One way to enhance the hybridization is exploitation of metal-ion mediated base-pairing, i. e. oligonucleotide probes that tightly bind a metal ions and are able to form a coordinative bonds between modified and natural nucleobases. This kind of metallo basepairs containing short modified oligonucleotides can also be useful for recognition of other RNA sequences containing hairpin-like structural motives, such as the TAR sequence of HIV. In addition, metal-ion-binding oligonucleotides will undoubtedly find applications in DNA-based nanotechnology. In this study, the 3,5-dimethylpyrazol-1-yl substituted purine derivatives were successfully incorporated within oligonucleotides, into either a terminal or non-terminal position. Among all of the modified oligonucleotides studied, a 2-(3,5-dimethylpyrazol-1-yl)-6-oxopurine base containing oligonucleotide was observed to bind most efficiently to their unmodified complementary sequences in the presence of both Cu2+ or Zn2+. The oligonucleotide incorporating 2,6-bis(3,5-dimethylpyrazol-1-yl)purine base also markedly increased the stability of duplexes in the presence of Cu2+ without losing the selectivity.

Recognition of nucleic acids by metal ion complexes

Metal-ion-mediated base-pairing of nucleic acids has attracted considerable attention during the past decade, since it offers means to expand the genetic code by artificial base-pairs, to create predesigned molecular architecture by metal-ion-mediated inter- or intra-strand cross-links, or to convert double stranded DNA to a nano-scale wire. Such applications largely depend on the presence of a modified nucleobase in both strands engaged in the duplex formation. Hybridization of metal-ion-binding oligonucleotide analogs with natural nucleic acid sequences has received much less attention in spite of obvious applications. While the natural oligonucleotides hybridize with high selectivity, their affinity for complementary sequences is inadequate for a number of applications. In the case of DNA, for example, more than 10 consecutive Watson-Crick base pairs are required for a stable duplex at room temperature, making targeting of sequences shorter than this challenging. For example, many types of cancer exhibit distinctive profiles of oncogenic miRNA, the diagnostics of which is, however, difficult owing to the presence of only short single stranded loop structures. Metallo-oligonucleotides, with their superior affinity towards their natural complements, would offer a way to overcome the low stability of short duplexes. In this study a number of metal-ion-binding surrogate nucleosides were prepared and their interaction with nucleoside 5´-monophosphates (NMPs) has been investigated by 1H NMR spectroscopy. To find metal ion complexes that could discriminate between natural nucleobases upon double helix formation, glycol nucleic acid (GNA) sequences carrying a PdII ion with vacant coordination sites at a predetermined position were synthesized and their affinity to complementary as well as mismatched counterparts quantified by UV-melting measurements.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Prediction of Phosphorylation Sites from Protein Sequences

Phosphorylation is amongst the most crucial and well-studied post-translational modifications. It is involved in multiple cellular processes which makes phosphorylation prediction vital for understanding protein functions. However, wet-lab techniques are labour and time intensive. Thus, computational tools are required for efficiency. This project aims to provide a novel way to predict phosphorylation sites from protein sequences by adding flexibility and Sezerman Grouping amino acid similarity measure to previous methods, as discovering new protein sequences happens at a greater rate than determining protein structures. The predictor – NOPAY - relies on Support Vector Machines (SVMs) for classification. The features include amino acid encoding, amino acid grouping, predicted secondary structure, predicted protein disorder, predicted protein flexibility, solvent accessibility, hydrophobicity and volume. As a result, we have managed to improve phosphorylation prediction accuracy for Homo sapiens by 3% and 6.1% for Mus musculus. Sensitivity at 99% specificity was also increased by 6% for Homo sapiens and for Mus musculus by 5% on independent test sets. In this study, we have managed to increase phosphorylation prediction accuracy for Homo sapiens and Mus musculus. When there is enough data, future versions of the software may also be able to predict other organisms.