Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin–biotin recognition

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin–biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics

Synthesis, Purification and Crystallization of Guanine-rich RNA Oligonucleotides

Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution

Interaction of Pyrrolobenzodiazepine (PBD) Ligands with Parallel Intermolecular G-Quadruplex Complex Using Spectroscopy and ESI-MS

Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5′GGGGTTGGGG3′) designated as d(T2G8), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T2G8) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T2G8)2 and d(T2G8)4 forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T2G8) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex

Interactions between metal ions and DNA

84 years elapsed between the announcements of the periodic table and that of the DNA double helix in 1953, and the two have been combined in many ways since then. In this chapter an outline of the fundamentals of DNA structure leads into a range of examples showing how the natural magnesium and potassium ions found in nature can be substituted in a diversity of applications. The dynamic structures found in nature have been studied in the more controlled but artificial environment of the DNA crystal using examples from sodium to platinum and also in a range of DNA-binding metal complexes. While NMR is an essential technique for studying nucleic acid structure and conformation, most of our knowledge of metal ion binding has come from X-ray crystallography. These days the structures studied, and therefore also the diversity of metal binding, go beyond the double helix to triplexes, hairpin loops, junctions and quadruplexes, and the chapter describes briefly how these pieces fit into the DNA jigsaw. In a final section, the roles of metal cations in the crystallisation of new DNA structures are discussed, along with an introduction to the versatility of the periodic table of absorption edges for nucleic acid structure determination

Staplerhämorrhoidopexie im Vergleich zur Milligan-Morgan-und Ferguson-Hämorrhoidektomie: ein systematisches Review

Fragestellung: Ziel dieser Studie war, ein systematisches Review und eine Metaanalyse der Kurz- und Langzeitergebnisse der Staplerhämorrhoidopexie durchzuführen. Patienten und Methodik: Mit einer Literatursuche wurden randomisierte, kontrollierte Studien zum Vergleich von Staplerhämorrhoidopexie und Milligan-Morgan-/ Ferguson-Hämorrhoidektomie abgefragt. Die Daten wurden für jede Studie einzeln entnommen und die Unterschiede mit Fixed- und Random-Effects-Modellen analysiert. Ergebnisse: Es wurden 34 randomisierte Studien und zwei systematische Reviews gefunden; hiervon wurden 29 Studien eingeschlossen. Die Staplerhämorrhoidopexie erwies sich in Bezug auf den Klinikaufenthalt (p < 0,001) als statistisch signifikant überlegen und hinsichtlich der postoperativen Schmerzen (perioperativ und früh-postoperativ), der Operationsdauer sowie der Blutungen (postoperativ und spät-postoperativ) als numerisch überlegen. Nach der Staplerhämorrhoidopexie waren Prolapsrezidive und wiederholte Eingriffe aufgrund von Rezidiven häufiger. Bei den Komplikationsraten wurden keine Unterschiede beobachtet. Schlussfolgerung: Die Staplerhämorrhoidopexie reduziert die Dauer des Krankenhausaufenthalts und könnte einen Vorteil im Sinne einer kürzeren Operationsdauer, weniger postoperativer Schmerzen und geringerer Blutungen bieten, ist jedoch mit einer erhöhten Rate von Prolapsrezidiven assoziiert

Staplerhämorrhoidopexie im Vergleich zur Milligan-Morgan-und Ferguson-Hämorrhoidektomie: ein systematisches Review

Fragestellung: Ziel dieser Studie war, ein systematisches Review und eine Metaanalyse der Kurz- und Langzeitergebnisse der Staplerhämorrhoidopexie durchzuführen. Patienten und Methodik: Mit einer Literatursuche wurden randomisierte, kontrollierte Studien zum Vergleich von Staplerhämorrhoidopexie und Milligan-Morgan-/ Ferguson-Hämorrhoidektomie abgefragt. Die Daten wurden für jede Studie einzeln entnommen und die Unterschiede mit Fixed- und Random-Effects-Modellen analysiert. Ergebnisse: Es wurden 34 randomisierte Studien und zwei systematische Reviews gefunden; hiervon wurden 29 Studien eingeschlossen. Die Staplerhämorrhoidopexie erwies sich in Bezug auf den Klinikaufenthalt (p < 0,001) als statistisch signifikant überlegen und hinsichtlich der postoperativen Schmerzen (perioperativ und früh-postoperativ), der Operationsdauer sowie der Blutungen (postoperativ und spät-postoperativ) als numerisch überlegen. Nach der Staplerhämorrhoidopexie waren Prolapsrezidive und wiederholte Eingriffe aufgrund von Rezidiven häufiger. Bei den Komplikationsraten wurden keine Unterschiede beobachtet. Schlussfolgerung: Die Staplerhämorrhoidopexie reduziert die Dauer des Krankenhausaufenthalts und könnte einen Vorteil im Sinne einer kürzeren Operationsdauer, weniger postoperativer Schmerzen und geringerer Blutungen bieten, ist jedoch mit einer erhöhten Rate von Prolapsrezidiven assoziiert

Stapled haemorrhoidopexy compared to Milligan–Morgan and Ferguson haemorrhoidectomy: a systematic review

Purpose The aim of this study was to perform a systematic review and meta-analysis of the short- and long-term outcomes of stapled haemorrhoidopexy.Methods A literature search identified randomised controlled trials comparing stapled haemorrhoidopexy with Milligan–Morgan/Ferguson haemorrhoidectomy. Data were extracted independently for each study and differences analysed with fixed and random effects models.Results Thirty-four randomised trials and two systematic reviews were identified, and 29 trials included. Stapled haemorrhoidopexy was statistically superior for hospital stay (p < 0.001) and numerically superior for post-operative pain (peri-operative and mid-term), operation time and bleeding (post-operative and long-term). Recurrent prolapse and re-intervention for recurrence were more frequent following stapled haemorrhoidopexy. No difference was observed in the rates of complications.Conclusions Stapled haemorrhoidopexy reduces the length of hospital stay and may have an advantage in terms of decreased operating time, reduced post-operative pain and less bleeding but is associated with an increased rate of recurrent prolapse

Stapled haemorrhoidopexy compared to Milligan–Morgan and Ferguson haemorrhoidectomy: a systematic review

Purpose The aim of this study was to perform a systematic review and meta-analysis of the short- and long-term outcomes of stapled haemorrhoidopexy.Methods A literature search identified randomised controlled trials comparing stapled haemorrhoidopexy with Milligan–Morgan/Ferguson haemorrhoidectomy. Data were extracted independently for each study and differences analysed with fixed and random effects models.Results Thirty-four randomised trials and two systematic reviews were identified, and 29 trials included. Stapled haemorrhoidopexy was statistically superior for hospital stay (p < 0.001) and numerically superior for post-operative pain (peri-operative and mid-term), operation time and bleeding (post-operative and long-term). Recurrent prolapse and re-intervention for recurrence were more frequent following stapled haemorrhoidopexy. No difference was observed in the rates of complications.Conclusions Stapled haemorrhoidopexy reduces the length of hospital stay and may have an advantage in terms of decreased operating time, reduced post-operative pain and less bleeding but is associated with an increased rate of recurrent prolapse