2′-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA

Abstract

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2′-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.

i-Motif DNA: structural features and significance to cell biology

“…Thus, i-motif structures were observed at neutral pH and low temperatures under molecular crowding conditions ( 18 , 19 ), under negative superhelicity ( 20 ), in the presence of silver ( 21 ) or copper (I) cations ( 22 ), and inside silica nano-channels ( 23 ). Chemical modifications such as 2′-deoxy-2′-fluoro-arabinocytidine (2′F-araC, known as 2′F-ANA) also induce formation of DNA i-motif structures under neutral conditions ( 24 , 25 ).…”

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

“…guanosines in the telomeric sequence AGGG(TTAGGG)3 with 2'F-araG leads to a 15°C 1 increase in Tm of the resulting intramolecular G4, and a shift from the usual antiparallel or 2 hybrid topology of this sequence (11,43) to a parallel conformation ( Figure 1a) (44). Here, we 3 demonstrate that G4 formed from the unmodified sequence (22G0) in KCl is a poor telomerase 4 substrate, leading to the previously-observed stuttering pattern (26) in a direct telomerase 5 extension assay involving incorporation of radiolabeled α 32 P-dGTP ( Figure 1b).…”

“…It has previously been difficult to distinguish whether 22 it is the parallel or intermolecular nature of G4 structures that allows their recognition by 23 telomerase. Here, we overcome this difficulty by exploiting the stabilization of intramolecular 24 parallel G4 conferred by the modified nucleotide 2'F-araG (44), enabling the demonstration 1 that inter-and intramolecular parallel G4 are extended by telomerase using the same 3-step 2 mechanism. These data reveal human telomerase to be a parallel G4 resolvase.…”

“…Oligonucleotides and G-quadruplex preparation 19 Most DNA oligonucleotides ( Supplementary Table 1) were purchased from Integrated DNA 20 Technologies and RNA oligonucleotides from Dharmacon, with purification by high 21 performance liquid chromatography (HPLC). Oligonucleotides 22G0, 22G3, and 22G0+tail 22 were synthesized as previously described (44). Synthesis of (C5-alkylamino)-dT-41G3 (the 23 equivalent of 22G3+tail prior to AlexaFluor 555 TM conjugation) was performed on an ABI 1 3400 DNA synthesizer (Applied Biosystems) at 1 μmol scale on Unylinker CPG solid support 2 (Chemgenes).…”

“…Synthesis of (C5-alkylamino)-dT-41G3 (the 23 equivalent of 22G3+tail prior to AlexaFluor 555 TM conjugation) was performed on an ABI 1 3400 DNA synthesizer (Applied Biosystems) at 1 μmol scale on Unylinker CPG solid support 2 (Chemgenes). Conjugation of (C5-alkylamino)-dT-41G3 to AlexaFluor 555 TM NHS Ester to 3 produce the desired 22G3+tail oligonucleotide was achieved following the standard protocol product was purified by anion exchange HPLC as described (44). The peak of (C5- Table 1) under the same 23 folding conditions as given for 22G0.…”