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G-kvadrupleksi, ki spadajo med nekanonične oblike DNA, imajo pomembno vlogo pri nadzorovanju stabilnosti genoma, regulaciji transkripcije in translacije. Nedavna bioinformatska analiza človeškega genoma je odkrila zaporedja oblike (G5/6H)3G5/6, ki bi lahko tvorila do zdaj še neopisane pet- oziroma šestkvartetne G-kvadruplekse. Z namenom, da bi določili vlogo in obliko posameznih elementov zaporedja ter njihov vpliv na topologijo G-kvadrupleksov, smo izhajali iz dobro raziskanega in podobnega zaporedja T(G3T)4 ter sistematično podaljševali dolžino G-traktov oziroma zank. Zvitje oligonukleotidov smo določali pri naraščajoči koncentraciji kalijevih ionov, po dvotedenski inkubaciji pri 4 °C, prileganju s počasnim ohlajanjem in prileganju s hitrim ohlajanjem z metodami jedrske magnetne resonance in poliakrilamidne gelske elektroforeze v nativnih pogojih. Medtem ko večina oligonukleotidov z G6-trakti tvori več struktur v raztopini, smo oligonukleotidom z G-trakti dolgimi tri, štiri in pet nukleotidov določili paralelno oziroma hibridno (3+1) topologijo. Možnost nastanka šestkvartetnega G-kvadrupleksa smo potrdili z zaporedjem TG6T, kjer topologije ne omejujeta dolžina in fleksibilnost zank in ki tvori paralelen tetrameren kompleks. Določitev topologij, ki prevladujejo pri zvitju v analiziranih pogojih, pomembno prispeva k raziskavam oligonukleotidnih zaporedij z G5/6-trakti. Pet- in šestkvartetni G-kvadrupleksi zaradi svoje redkosti v genomu predstavljajo potencialno specifično tarčo za zdravljenje rakavih obolenj in nevrodegenerativnih bolezni. One of the most well-researched forms of noncanonical DNA are G-quadruplexes, an important regulator of genome stability, transcription, and translation. Recent bioinformatic analyses of the human genome have discovered oligonucleotide sequences with the potential to form five and six stacked G-quadruplexes that have not been observed yet. We investigated oligonucleotide topologies through means of magnetic resonance spectroscopy and polyacrylamide gel electrophoresis. The topology of oligonucleotides with five and six G-tracts was studied with gradual G-tract and loop elongation of a well-characterised oligonucleotide sequence T(G3T)4. The folding patterns were investigated with increasing concentration of potassium cations, after annealing, quenching, and two-week-long incubation at 4 °C. Most samples displayed characteristics of structural polymorphism. Oligonucleotides with G-tract length of three, four and five nucleotides formed predominantly one structure, which allowed us to determine two parallel and one hybrid (3+1) topology, respectively. We also confirmed the existence of six stacked G-quadruplex in a form of a parallel tetrameric complex. The discovery of structures that preferentially form under analysed conditions facilitates further research into five or six stacked G-quadruplexes. Studies may continue with the examination of a variety of loop types, as such G-quadruplexes may prove to be an important therapeutical target in the future. |
