The Importance of Phosphates for DNA G‐Quadruplex Formation: Evaluation of Zwitterionic G‐Rich Oligodeoxynucleotides

Abstract

A quaternary ammonium butylsulfonyl phosphoramidate group (N+) was designed to replace all the phosphates in a G‐rich oligodeoxynucleotide d(TG4T), resulting in a formally charge‐neutral zwitterionic N+TG4T sequence. We evaluated the effects of N+phosphate modifications on the structural, thermodynamic and kinetic properties of the parallel G‐quadruplexes (G4) formed by TG4T and compared them to the properties of the recently published phosphoryl guanidine d(TG4T) (PG‐TG4T). Using size‐exclusion chromatography, we established that, unlike PG‐TG4T, which exists as a mixture of complexes of different molecularity in solution, N+TG4T forms an individual tetramolecular complex. In contrast to PG modifications that destabilized G4s, the presence of N+ modifications increased thermal stability relative to unmodified [d(TG4T)]4. The initial stage of assembly of N+TG4T proceeded faster in the presence of Na+ than K+ions and, similarly to PG‐TG4T, was independent of the salt concentration. However, after complex formation exceeded 75 %, N+TG4T in solution with Na+showed slower association than with K+. N+TG4T could also form G4s in solution with Li+ions at a very low strand concentration (10 μM); something that has never been reported for the native d(TG4T). Charge‐neutral PG‐G4s can invade preformed native G4s, whereas no invasion was observed between N+and native G4s, possibly due to the increased thermal stability of [N+TG4T]4. The N+ modification makes d(TG4T) fully resistant to enzymatic digestion, which could be useful for intracellular application of N+‐modified DNA or RNA.

DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies

“…Synthesis and purification of modified ONs 4-(Azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide [38] and tosyl azide (p-toluenesulfonyl azide, TsN 3 ) [39] were synthesised and used for the synthesis of the modified ONs using an automated DNA synthesiser as described. The solution of sulfonyl azide (0.5 M TsN 3 in MeCN or 0.7 M 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide in DMF) was 6− ; e synthesised in a 1 µmol scale using a previously reported procedure with transferring the solid support from a column into a vial for reaction with sulfonyl azide for 30 min at room temperature.…”

“…Some amount of the solid support was lost during the transfer and washing steps, especially for multiple modifications, which was the main reason for the low yields of these ONs. f synthesised in a 1 µmol scale following a modified procedure using a microtube pump to deliver the sulfonyl azide solution onto the column with CPG-support at 37 °C [38]; g synthesised in a 3-4 µmol scale, purified by 20% denaturing PAGE (7 M urea), followed by extraction from the gel and desalting. introduced as replacement of a standard iodine/pyridine oxidation step to react with 3',5'-dinucleoside β-cyanoethyl phosphites (Scheme 1, I), forming the N-modified iminophosphorane (Scheme 1, II).…”

“…Similarly, the incorporation of BCNS groups on a DNA backbone requires the synthesis of thymidine and 2'-OMe-uridine phosphoramidites bearing BCNS groups comprising nine and ten synthetic steps, respectively. In comparison, the synthesis of an N+ monomer requires only four synthetic steps starting from commercially available 1,4-butane sultone that does not have any silica gel purification [38]. The incorporation of the N+ modification onto DNA is performed during DNA synthesis instead of a standard oxidation step.…”

“…For example, 92.0 ± 1.8% of 4N+ON7 remained intact, whereas only 54 ± 3% of 4Ts-ON13 was intact after 120 min of enzymatic digestion (Figure 2, see also Figure S17 in Supporting Information File 1). N+ONs possessing more than four modifications showed a full enzymatic resistance after 120 min [38].…”

“…Recently, we synthesised a G-rich ON (TG 4 T) with all phosphates replaced by a 4-(trimethylammonio)butylsulfonyl phosphoramidate group (N+, Scheme 1). The sequence was designed to obtain the formally charge-neutral zwitterionic N+TG 4 T [38]. Each negatively charged phosphoramidate is neutralised by the positively charged quaternary ammonium group, providing a zwitterionic phosphate mimic.…”