Electron paramagnetic resonance (EPR) spectroscopy is a well known technique that is routinely used to investigate the structure and dynamics of nucleic acids. EPR requires incorporation of paramagnetic centers at specific sites, called site-directed spin labeling (SDSL). Spin labeling of nucleic acids can be carried out by two approaches, either by forming a covalent bond between spin label and the nucleic acid or by noncovalent binding of the spin label to the nucleic acid. This PhD dissertation is focused on advancing noncovalent and site-directed spin labeling (NC-SDSL) of nucleic acids. Our approach is based on ligand-receptor interactions, where the spin label binds to an abasic site of nucleic acids through formation of Watson-Crick base pairing with an orphan base on the opposing strand and pi- stacking interaction.
A series of purine-based spin labels were prepared. Both 1,1,3,3- tetramethylisoindolin-2-yloxyl and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were conjugated with either the C2- or C6-position of the purines, yielding nitroxide derivatives of guanine, adenine or 2,6-diaminopurine. The binding of these spin- labeled purines to abasic sites in duplex nucleic acids was evaluated by EPR spectroscopy. The isoindoline-derived spin labels showed full binding to abasic sites in RNA duplexes and emerged as superior binders when compared to TEMPO-derived spin labels. The guanine-derived Ǵ bound fully to both DNA and RNA duplexes at lower temperature when paired with cytosine (C). Ǵ bound much more efficiently to RNA than DNA and showed even extensive binding at 20 °C (Kd = 6.15 x 10-6 M). Adenine-derived spin label 28 was also bound fully at low temperature (-30 °C) to abasic sites in both DNA and RNA duplexes when paired with T and U, respectively, but showed only minor binding at 20 °C (< 5%). Thus,
Ǵ and 28 allow noncovalent and site directed spin labeling of abasic sites in nucleic acids, using any of the pyrimidines as orphan bases on the opposing strand.
The spin label that bound with the highest efficiency (Ǵ) was chosen to investigate further various aspects of noncovalent spin labeling. Ǵ was found to bind to abasic sites in DNA/RNA hybrids, either to the DNA- or the RNA-strand. Ǵ showed only a minor flanking sequence effect upon binding to abasic sites in RNA, with binding affinities within a factor of two of each other. For a given base on the 5’-end, the highest binding was observed when a U was on the 3’-side. When the abasic site was placed close to the duplex end, the affinity of the spin label was reduced, presumably due to the increased flexibility at base pairs in the vicinity of the duplex terminus. Abasic sites in the fourth position from the duplex end had the same affinity as abasic sites towards the middle of the duplex.
A distance measurement between two spin labels was performed on a Ǵ- labeled 22-mer RNA duplex using pulsed electron-electron double resonance (PELDOR) spectroscopy in collaboration with Prof. Thomas Prisner (Goethe University, Germany). The PELDOR data showed a strong orientation dependence and a distance of 31.3 ± 3.5 Å, similar to the distance of ca. 29 Å, obtained by molecular modelling.
The noncovalent binding of purine-derived spin labels to the DNA triplex containing one nucleotide gap as binding site in phosphate- as well as magnesium containing-buffer was also evaluated by EPR spectroscopy. The nitroxide connected to N6-position of purine (28-31) had less binding to triplex DNA (IV-VI), whereas Ǵ bound almost fully to the triplex DNA V and VI in magnesium containing buffer. This project was pursued in collaboration with Prof. Clemens Richert’s research group at University of Stuttgart, Germany who provided the triplex DNA motifs.
Rafeindaspunalitrófsgreining (e. electron paramagnetic resonance (EPR) spectroscopy) er vel þekkt aðferð sem er reglubundið beitt í rannsóknum á byggingu og hreyfingu kjarnsýra. EPR mælingar grundvallast á því að koma meðseglandi kjarna fyrir á ákveðnum stað innan kjarnsýra, þ. e. staðbundnum spunamerkingum (e. site directed spin-labeling, SDSL). Spunamerkingaraðferðin sem hér er lýst byggist á víxlverkun milli tengils og viðtaka, þar sem spunamerkið binst við basalausa stöðu (basaeyðu) í tvíþátta kjarnsýrum, annars vegar með Watson-Crick basapörun við staka basann á mótstæða strendingnum og hins vegar með pí-stöflun við aðliggjandi basa. Safn púrín-tengdra spunamerkja var útbúið með bæði 1,1,3,3-tetrametýlísóindólín-2-ýloxýl og 2,2,6,6-tetrametýlpiperidín-N-oxýl (TEMPO) merkihópum, sem voru tengdir inn á C2- eða C6-stöðuna á púríni. Þannig fengust nítroxíð afleiður gúaníns, adeníns eða 2,6-díamínópúríns. Tengigeta þessara spunamerktu púrína við basaeyðu í tvíþátta kjarnsýrum var mæld með EPR mælingu. Afleiður ísóindólín spunamerkjanna tengdust fullkomlega við slíkar eyður í tvíþátta RNA og mun betur en TEMPO afleiðurnar. Gúanín afleiðan Ǵ tengdist að fullu við bæði tvíþátta DNA og RNA við lágt hitastig, þegar hún var pöruð við sýtósín (C). Ǵ tengdist betur við RNA en DNA og sýndi jafnvel mikla bindingu við 20 °C (Kd = 6,15∙10-6 M). Adenín-afleidda spunamerkið 5 sýndi einnig hámarks tengingu við lágt hitastig (-30 °C) við basaeyðuna á tvíþátta DNA og RNA á móti T annars vegar og U hins vegar. Spunamerkið Ǵ var síðan valið til að rannsaka nánar ýmsa þætti spunamerkingar án samgildra tengja. Í ljós kom að Ǵ tengdist basaeyðum í DNA/RNA blendingum, annaðhvort við DNA- eða RNA strendinginn. Basarnir við hliðina á basalausu stöðunni höfðu aðeins lítil áhrif á tengingu Ǵ við eyðuna í RNA. Þegar slík eyða var staðsett nálægt endandum á tvíþátta kjarnsýrunni, minnkaði sækni spunamerkisins, væntanlega vegna aukins hreyfanleika basapara sem staðsett eru í nánd við endann. Fjarlægðamæling milli tveggja spunamerkja var framkvæmd á Ǵ-merktu tvíþátta RNA með notkun PELDOR (e. pulsed electron-electron double resonance) í samvinnu við Prof. Thomas Prisner við Goethe Háskólann í Frankfurt, Þýskalandi. PELDOR mælingarnar voru háðar innbyrðis afstöðu (e. orientation dependence) spunamerkjanna og gáfu fjarlægðina 31,3 ± 3,5 Å, sem reyndist í góðu samræmi við fjarlægðina í líkönum (29 Å).
