Na⁺ selective structural switch from an intramolecular triplex to tetrad stabilised by non-canonical mispairs in double repeat of Arabidopsis thaliana telomere (T₃AG₃)₂

Abstract

DNA is polymorphic, as with four nucleobases, it can be configured in a number of secondary structures. The four-stranded DNA structures consisting of G-tetrads have especially been intriguing because of their proven existence in human cells. Due to the high prevalence of putative G-quadruplex-forming sequence motifs in the human genome, scientists in recent years have highlighted the potential of exploiting these exotic structures for targeted therapies for various cancers. G-quadruplexes are the most common and well-studied arrangements of four guanines; however, other possible non-canonical arrangements of nucleobases have also been reported. Herein, using Gel electrophoresis, Circular Dichroism, UV & CD-thermal denaturation methods, and NMR, we suggested that a double repeat of Arabidopsis thaliana telomere (T₃AG₃)₂ shows a structural switch from a non-canonical intramolecular triplex to a non-conventional tetrad other than an antiparallel G-quadruplex. This transition is mediated by increasing Na⁺ cation concentration from 0.1 M to 1.0 M, and the tetrad is fairly stabilised by a hydrogen-bonded cyclic array of non-canonical/mismatch base pairs (G∗G, G∗T, and T∗T). Intriguingly, such a structural transition was not manifested in the presence of K⁺ ions. To the best of our knowledge, such a cation-specific non-canonical structural switch, in a telomeric sequence, has not been proposed to date.

Highlights

• •Arabidopsis thaliana telomere shows structural transition from mini-triplex to a quadruplex as a function of Na⁺ ion concentration.
• •Structures are stabilised by mismatch base pairs (G∗G, G∗T, and T∗T).
• •A modest attempt to show that structural topology of telomeric sequence is no more conserved as G-quartet.

1. Introduction

G-quadruplexes are higher-order non-canonical nucleic acid structures formed by guanine–rich strands. The four guanines constitute a square planar structure where each guanine serves as both a hydrogen bond donor and an acceptor and thus forming a tetrad of Hoogsteen hydrogen-bonded guanines. The stability of these tetrads is ruled by eight hydrogen bonds and the electrostatic interaction with cations present between two consecutive tetrads. Numerous studies have documented the order of ions to determine the stability of G-quadruplexes; however, the physiologically relevant Na⁺ and K⁺ have been found to play a significant role [[1], [2], [3]].

Needless to say, G-quadruplexes are the most studied tetrads, exhibiting wide structural diversity regarding strand orientation, connecting loop topologies, cations, etc. [4,5]. Nevertheless, as per the documented literature, this is not the only possible arrangement of four nucleobases. Formation of mixed tetrads by the sequences with nucleobases other than G_n repeats has dramatically increased the versatility of quadruplex formation [2,6,7]. Also, literature is rich in databases like DSSR-G4DB (http://g4.x3dna.org) and the ONQUADRO database, which collects tetrads, quadruplexes, and G4-helices found in PDB-deposited structures of nucleic acids. Plenty of information like sequences, secondary and tertiary structures, and motif-specific description including planarity, rise and twist parameters, Webba da Silva geometric formalism, etc., is stored there [8,9].

Escaja et al. have nicely reviewed the topological diversity of experimentally proved tetrads like homo-tetrads, base pair tetrads, minor groove tetrads, major groove tetrads, etc. The stability of such tetrads has been an interesting aspect of investigation [2]. For instance, Caceres et al., using single crystal X-ray diffraction, demonstrated the formation of thymine tetrad intervening guanine tetrads by the sequence d(TGGGT)₄. It has been shown that a central sodium ion interacting with two thymines contributes to the tetrad structure. The tetrad structures are well stabilised by Na⁺/Tl⁺ ions [10]. In light of these supporting pieces of evidence, we have experimentally demonstrated here the formation of a novel mixed tetrad at higher Na⁺ ion concentration in the double repeats of Arabidopsis thaliana telomere (T₃AG₃)₂ (Arab2), while the 3.5 repeats of the same telomeric sequence [(GGG(TTTAGGG)₃] has been found to fold into conventional G-quadruplex where mixed antiparallel and parallel structure are displayed in the presence of potassium ion while antiparallel topology is obtained in the presence of sodium ion. Also, no structure could be depicted in the presence of lithium ion [11].

Arabidopsis thaliana is a small, short-lived flowering plant with a relatively small genome of approximately 135 Mbp and five chromosomes. It was the first plant to have its genome sequenced and is considered a model organism to understand plant biology and genetics [12,13]. Being a halophyte, A. thaliana can thrive in high salt concentration identifying it's salt-adaptive mechanisms. The salt-sensitive nature of Arabidopsis could be a genetic adaptation that enables a halophytic lifestyle, enabling functions of genes to facilitate adaptation to saline conditions. This has great potential for improving the tolerance of crop plants to salinized soils [14].

The present study is a modest attempt to identify and characterize the structural topology of a double repeat of A. thaliana telomere (T₃AG₃)₂ in the presence of Na⁺ using a combination of techniques, as gel electrophoresis, Circular dichroism, UV & CD-thermal melting, and NMR. We proposed here an unusual Na⁺ concentration-dependent structural shift from a non-canonical intramolecular mini-triplex to a non-conventional tetrad structure.

2. Materials and methods

The PAGE-purified oligonucleotides were purchased on a 1 μM scale from Bio Basic Inc., Canada, for biophysical studies. The concentration of the oligonucleotides was determined spectrophotometrically by using the extinction coefficient (ε) calculated by the nearest neighbor method and measuring the absorbance at 260 nm. Stock solutions of the oligomers were prepared by directly dissolving the lyophilized powder in MilliQ water. The different oligonucleotide sequences used for the study are listed below.

S.No.	Name	Oligonucleotide Sequence	ε(M−1 cm−1)
1	Arab2	5′- TTT AGG GTT TAG GG-3′	138, 600
2	Arab4	5′- TTT AGG GTT TAG GGT TTA GGG TTT AGG G -3′	277, 000
3	CArab2	5′- CCC TAA ACC CTA AA -3′	135, 000
4	CArab4	5′- CCC TAA ACC CTA AAC CCT AAA CCC TAA A -3′	268, 400
5	PAL12	5'- CTT GAG CTC AAG -3'	113, 700

2.1. Non- denaturing gel electrophoresis

Non-denaturing, 20% PAGE was used to study the size and molecularity of the oligonucleotides under study. Both the gel and 20 μM oligomer-containing samples contained 20 mM sodium cacodylate pH = 7.4, 0.1 mM EDTA, and 0.1 M of NaCl, whereas the running buffer contained 1X TBE, 0.1 mM EDTA, and 0.1 M salt. The samples were heated at 100 °C in the water bath for 5 min, and then they were allowed to cool slowly at room temperature for annealing, and then we kept them overnight at 4 °C for incubation before loading into the gel. Orange-G mixed with glycerol was used as a tracking dye, and Stains-all was used for staining the bands after electrophoresis, finally visualized under white light and photographed by AlphalmagerTM-2200 (Alpha Infotech Corp.).

2.2. Denaturing gel electrophoresis

A 20% denaturing polyacrylamide solution containing 7 M urea was used to cast the gel matrix. The samples were prepared by mixing 10 μM oligonucleotide and 1X TBE in 100% formamide, and the running buffer contained 1X TBE. The samples were heated at 100 °C for 5 min, then were given a cold shock for 15 min. The samples were loaded into the gel after mixing with tracking dye, which is Orange-G mixed with glycerol. Finally, the bands were visualized after staining with Stains all under white light and photographed by AlphalmagerTM-2200 (Alpha Infotech Corp.).

2.3. UV-thermal denaturation

UV-melting curves were recorded from 10 °C to 95 °C at 295 nm with a heating rate of 0.5 °C/min using the stoppered quartz cells of 10 mm and 1 mm path length with a volume capacity of 110 μL and 35 μL, respectively. The thermal melting spectra were registered on UV-1650 PC Shimadzu UV-vis spectrophotometer equipped with a Peltier thermo-programmer, TMS PC-8(E)-200. The samples contained 20 mM sodium cacodylate pH = 7.4, 0.1 mM EDTA, and 0.1 M of NaCl and different concentration of Arab2 ranges from 10 μM to 50 μM for studying oligomer concentration dependence and for salt dependence, the samples contained 20 mM sodium cacodylate pH = 7.4, 0.1 mM EDTA, 5 μM Arab2 and different concentration of NaCl ranges from 0.1 M − 1 M. The samples were annealed and incubated in the same manner as for non-denaturing gel electrophoresis.

2.4. Circular dichroism

CD spectra were recorded with a scanning speed of 100 nm/min and collected on a JASCO-815 spectrophotometer interfaced with an IBM PC compatible computer at 20 °C, as an average of three scans between the ranges of 200–350 nm using quartz cells of 10 mm path length with a volume of 1 mL.

For CD melting experiments, CD measurements were performed at 280 nm and 295 nm wavelengths with a heating rate of 1 °C/min for the temperature range from 5 °C - 95 °C.

For both CD spectra and CD melting studies, the samples were prepared in 20 mM sodium cacodylate(pH = 7.4), 0.1 M EDTA, and different Na⁺ ion concentrations as required. The samples were annealed and incubated in the same manner as for other assays, like non-denaturing gel electrophoresis and UV-thermal melting.

2.5. Nuclear magnetic resonance

For the NMR experiment, Arab2 (T₃AG₃)₂ was commercially procured (synthesized on a 10 μM scale) and supplied as a gel-purified sample. The lyophilized Arab2 oligonucleotide was dissolved in 90% H₂O + 10% D₂O solvent containing 20 mM sodium cacodylate (pH 7.4) and 0.1 M NaCl in a total volume of 400 μL. ¹H NMR spectra were acquired at 20 °C on a Bruker Avance 500 MHz FT-NMR spectrometer at the NMR facility of the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi. For information on exchangeable protons, the 1D spectrum was recorded. Typical parameters for the 1D NMR with the pulse program were the number of data points = 16k, spectral width = 20 ppm, number of scans = 512, relaxation delay = 1.0 s, and pulse width = 15 μs.

3. Results and discussion

3.1. Non- denaturing gel electrophoresis

To obtain the structural status and ion-dependent changes in (T₃AG₃)₂ [Arab2], gel electrophoresis was performed in non-denaturing conditions. Fig. 1a presents the 20 % PAGE pattern of Arab2 at 20 μM oligomer concentration in 20 mM sodium cacodylate buffer (pH 7.4), containing 0.1 mM EDTA and 0.1, 0.5 M NaCl (lane 2, and 3). Other oligomers (used as control markers) loaded in lanes 1, 4, 5, and 6 comprise (CGCGAATTCGCG) PAL12, cArab2 (C₃TA₃)₂, the perfect duplexes of Arab2 [(T₃AG₃)₂ + (C₃TA₃)₂] and Arab4 [(T₃AG₃)₄+ (C₃TA₃)₄], respectively. These markers were used to compare the relative mobility of the bands produced by (T₃AG₃)₂ [Arab2] sequence under study. The gel analysis showed that the single band of Arab2 moves faster than PAL12 (lane 1), perfect duplexes of Arab2 and Arab4 (lanes 5and 6). The 12-nt long perfect palindromic sequence PAL moves as 12-mer duplex (24-nt) in native conditions, and the mobility of the duplex of Arab2 is equivalent to 14-mer duplex (28-nt) The higher mobility of Arab2, a 14-mer long sequence from PAL and its own duplex, completely rule out the possibility of its existence as a dimeric structure. Consequently, an unimolecular structure was speculated. The retarded mobility of Arab2 (14-mer, structured sequence) from its complementary sequence cArab2 (14-mer, unstructured) is attributed to the presence of thymine and guanine, as already been explained in another finding of unimolecular triple helical structure for double repeats of C. reinhardtii telomere [15].

Fig. 1a.

20% Native PAGE mobility pattern of oligonucleotide sequences. (a) lane 1: PAL12, lane 2,3: (T₃AG₃)₂[Arab2] in increasing concentrations of NaCl (0.1, 0.5 M), lane 4: CArab2, lane 5: (T₃AG₃)₂[Arab2] + (C₃TA₃)₂[CArab2], lane 6: (T₃AG₃)₄[Arab4] + (C₃TA₃)₄[CArab4] at 1:1 in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA.

Also, to further explain the retarded mobility of Arab2, double and four repeats of a few other telomeric sequences (Giardia lamblia [(TAG₃)₂ Gia2 and (TAG₃)₄ Gia4], Chlamydomonas reinhardtii [(T₄AG₃)₂ Chlm2 and (T₄AG₃)₄ Chlm4]) along with their complementary sequences [(C₃TA)₂-(CGia2), (C₃TA)₄-(CGia4) (C₃TA₄)₂ -CChlm2, (C₃TA₄)₄-CChlm4] were allowed to run in denaturing gel (Fig. 1b). Interestingly, the mobility pattern of all telomeric sequences was found to be retarded when compared with their respective complementary sequence. Therefore, the unimolecular status of the Arab2 is well supported by both native and denaturing gel electrophoresis.

Fig. 1b.

20% Denaturating PAGE mobility pattern of oligonucleotide sequences, Lane 1: (TAG₃)₂ Gia2; Lane 2: (C₃TA)₂ CGia2; Lane 3: (T₃AG₃)₂ Arab2; Lane 4: (C₃TA₃)₂ CArab2; Lane 5: (T₄AG₃)₂ Chlm2; Lane 6: (C₃TA₄)2 CChlm2; Lane 7: (TAG₃)₄ Gia4; Lane 8: (C₃CCTA)₄ CGia4; Lane 9: (T₃AG₃GG)₄ Arab4; Lane 10: (C₃TA₃)₂ CArab4.

The structures adopted by the double repeat (Arab2) of Arabidopsis thaliana telomere, detected by gel studies, highlighted their tendency to fold back, which, in order, is credited to the presence of four nucleotide base residues between each guanine stretch. The effect of loop length on the folding and stability of G-quadruplexes is well reported [16]. To define the complete status of (T₃AG₃)₂ in terms of strand stoichiometry, strand polarity, and stability, CD studies and UV thermal denaturation studies were also performed and are discussed in the following sections.

3.2. Circular dichroism

CD spectra of (T₃AG₃)₂ [Arab2] at 8 μM strand concentration in 20 mM sodium cacodylate (pH 7.4), and 0.1 mM EDTA at different NaCl concentrations (0.1 M – 1.0 M) are shown in Fig. 2. The spectra of (T₃AG₃)₂ in the presence of 0.1 M Na⁺ is characterized by a negative peak at 240 nm followed by a positive peak at 255 nm along with a small hump at 280 nm which on increasing the salt concentration to 1 M Na⁺, displayed a negative peak at 260 nm followed by a positive peak at 290 nm. The CD characteristics of Arab2 in the presence of 0.1 M Na⁺ are also at 255 nm and 280 nm, like we obtained in our earlier finding of intramolecular triple helical structure with double repeats of Chlm2 (T₄AG₃)₂, which differed in terms of ellipticities but displayed occurrence of G∗G and G∗T bonding [15]. Based on the facts discussed in our previous report, the CD bands obtained here at 0.1 M Na⁺ in the case of Arab2 symbolize an unimolecular structure (as shown in gel studies) stabilised by G∗G and G∗T bonding. Interestingly, it was found that with increasing concentration of Na⁺ ion, the negative peak at 240 nm gradually disappeared and a new negative peak at 260 nm developed, giving an isodichroic point at 250 nm. Also, the positive band at 255 nm and 280 nm has now been shifted to 290 nm. These CD signatures for antiparallel tetrad, i.e., 260 nm negative band followed by 290 nm positive band, are well supported by Randazzo et al., where the authors have elegantly explained the CD characteristics of G-quadruplexes, with different folding topologies [17]. It is explained that a positive band at 240 nm coupled with a negative band at 260 nm shows the absence of stacking bases of the same glycosidic bond angle (GBA) in the quadruplex stem. Also, a band at 290 nm is characteristic of the presence of stacking of nucleobases of different GBA (i.e., syn-anti, not syn-syn).

Fig. 2.

CD spectra of (T₃AG₃)₂ [Arab2] at 8 $\mu$ M strand concentration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA in increasing salt (NaCl) concentration.

Furthermore, the transition at 250 nm shows the disposition of two adjacent quartets in H-to-T orientation. Consequently, all these characteristics are in good agreement with the CD signatures we experienced at 1 M Na⁺ [17]. This illustrated that the monomeric structure adopted by (T₃AG₃)₂ [Arab2], most possibly stabilised by G∗G and G∗T base-mispair bonding at 0.1 M Na⁺ gets converted to an antiparallel tetrad, intertwined through T∗G∗G∗T quartets facilitated at 1.0 M Na⁺ (Fig. 2). Captivatingly, this specific shifting of CD characteristics was observed in the case of a double repeat of Arabidopsis thaliana telomere, specifically in the presence of Na⁺.

However, to show that the structural transitions from intramolecular triplex to tetrad are a specific case of double repeat of TTTAGGG as a function of sodium ion concentration, the status of double repeat of Arab as a function of K⁺ concentration and four repeat of Arab as a function of both Na⁺ and K⁺ concentration was also studied(Fig. 3(a and b,c)). In the presence of 0.1 M K⁺, Arab2 showed a negative peak at 240 nm followed by a positive peak at 260 nm, a characteristic of a parallel quadruplex. Increasing the K⁺ concentration from 0.1 M to 1 M did not show any significant transition of CD peak, except a minor shift of 15–20 nm, converting a sharp peak into a broad peak displaying the existence of both parallel and antiparallel conformation. Furthermore, such an ion dependence was investigated in the case of four repeats of Arabidopsis thaliana telomere, in Na⁺ and K⁺, each at 0.1 M or 1 M concentrations (Fig. 3(b and c)). In the presence of 0.1 M and 1 M Na⁺, both the four repeats of Arab (Arab4) displayed an identical CD spectrum characterized by a 260 nm negative peak followed by a positive peak at 295 nm, a typical signature of antiparallel quadruplex. Like Arab2 in the presence of K⁺, Arab4 also showed no topological transition as a function of K⁺ concentration. At 0.1 M and 1 M K⁺ ion concentration, both parallel and antiparallel conformations exist, depicted by 240 nm and positive peak splits into a hump at 265 nm and 292 nm. Thus, we propose here a structural transition of a double repeat TTTAGGG, as a function of sodium ion concentration, from an intramolecular triplex to a tetrad.

Fig. 3.

Fig. 3a: CD spectra of (T₃AG₃)₂ [Arab2] at 8 $\mathrm{\mu }$M strand concentration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, containing 0.1 M and 1 M KCl.

Fig. 3b: CD spectra of (T₃AG₃)₄ [Arab4] at 8 $\mathrm{\mu }$M strand concentration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, containing 0.1 M and 1 M NaCl.

Fig. 3c: CD spectra of (T₃AG₃)₄ [Arab4] at 8 $\mathrm{\mu }$M strand concentration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, containing 0.1 M and 1 M KCl.

To this point, the native gel and CD studies on the (T₃AG₃)₂ [Arab2] sequence revealed its existence as an intramolecular structure at a low salt concentration (0.1 M Na⁺), switching to an antiparallel tetrad at high salt concentration (1 M Na⁺). Further, for information on the thermal stability of the (T₃AG₃)₂ [Arab2] structure, UV-thermal denaturation studies were carried out.

3.3. UV thermal denaturation studies

Representative melting profiles of (T₃AG₃)₂ [Arab2] in 20 mM sodium cacodylate (pH 7.4) containing varied concentrations of Na⁺ (0.1, 0.3, 0.5, 1 M Na⁺) are shown in (Fig. 4a). The melting curves were monitored at 295 nm and were found to be of inverted monophasic nature in the presence of Na⁺ ions, demonstrating the melting of a single self-associated non-canonical structural form adopted by (T₃AG₃)₂ [Arab2]. Given the conclusions drawn from the gel and CD experiments, the observed melting behaviour of (T₃AG₃)₂[Arab2] in Na⁺ was as anticipated and went well with the interpretations. The Tm values calculated from the first derivative of the melting profiles came out to be 30 °C at 0.1 M Na⁺ concentration, which further increased to 41 °C at 1 M Na⁺ (Fig. 3). Going with the earlier work from the author's lab [15], the low stability of the oligomeric structure, even at 1 M salt concentration, was credited to G∗T wobble base pairs. The self-associated forms containing wobble base pairing are less stable than the standard Watson-Crick-Franklin base pairs [15].

Fig. 4a.

Thermal denaturation profile of (T₃AG₃)₂ [Arab2] at 5 $\mathrm{\mu }$M strand concentration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, containing 0.1 M, 0.3 M, 0.5 M, and 1 M NaCl, monitored at 295 nm.

In addition, to confirm the unimolecular status of the structural form of Arab2, the oligomer concentration-dependent UV-thermal spectra were monitored at 295 nm. The melting profile of Arab2 in 20 mM sodium cacodylate buffer (pH = 7.4), 0.1 mM EDTA, and 0.1 M NaCl, at varied oligonucleotide strand concentrations ranging from 10 μM to 50 μM displayed a monophasic inverted melting profile displaying a constant Tm value of 30 °C (Fig. 4b). This observation again strengthens our finding of an unimolecular (intramolecular) structure adopted by double repeats of A. thaliana telomere.

Fig. 4b.

Thermal denaturation profiles of (T₃AG₃)₂ [Arab2] in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA containing 0.1 M NaCl at 10 $\mathrm{\mu }$M, 20 $\mathrm{\mu }$M, 30 $\mathrm{\mu }$M, and 50 $\mathrm{\mu }$M strand concentration.

3.4. CD melting studies

Next, to confirm the observed structural and conformation switching at varied salt concentrations, the CD melting of Arab2 at 280 nm and 295 nm was carried out (Fig. 5). An interesting observation made from both the CD melting profiles proved another high point to reinforce our finding of a structural switch from the intramolecular triplex topology to the intramolecular tetrad, on increasing the salt concentration from 0.1 M to 1.0 M Na⁺ ion concentration respectively. It seems conceivable that the structures are most possibly stabilised by G∗G, G∗T, and T∗T bonding. As discussed in the author's earlier report [15] a proper melting profile at 255 nm couldn't be obtained due to the presence of mixed signatures of G∗G and G∗T bonding at ∼255 nm. Hence, CD melting profiles were recorded at 280 nm and 295 nm only. With identical Tm values, a significant difference in the ellipticity at 280 nm and 295 nm was observed at 0.1 M Na⁺, indicating that at 0.1 M salt, the structural topology existing at 280 nm has almost disappeared at 295 nm. The structure starts switching into a new topology, significant at 1.0 M salt concentration.

Fig. 5.

CD melting profiles of (T₃AG₃)₂[Arab2] at 5 $\mathrm{\mu }$M strand concentration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, containing (1) 0.1 M and (2) 1 M NaCl, monitored at 280 nm and 295 nm.

Worth mentioning is that at 1.0 M salt concentration, the high ellipticity at both 280 nm and 295 nm indicates the stabilization of intramolecular structure with the same G∗G and G∗T bonding patterns at 280 nm; however, in the presence of high salt, their folding pattern changed to a tetrad, which is reflected at 295 nm. The same effect was almost negligible at 0.1 M salt. In other words, if we consider the ellipticities of both the curves in the presence of 0.1 M and 1.0 M salt independently at 280 nm and 295 nm, we can observe that higher ellipticities at both salt concentrations at 280 nm signify the presence of possible G∗G and G∗T bonding, which gets further stabilised at higher salt. Further, the difference in ellipticities at 295 nm is attributed to the folding of Arab2 in the form of a tetrad topology, stabilised at high salt concentration.

3.5. NMR Studies

To further stamp the proposed intramolecular structure formed by Arab2, the NMR technique was used. It is well known that hydrogen-bonded imino proton resonances scatter over an appreciable spectral region, i.e, 12.5–16 ppm. The imino proton resonances of standard Watson-Crick- Franklin A•T base pairs are found between 13.0 and 14.5 ppm, while those of G•C base pairs occur between 11.5 and 13.5 ppm. Imino protons involved in the G-quadruplex Hoogsteen G∗G base pairing are observed in the region of 10–12 ppm [18]. While a set of exchangeable imino protons from the mismatched sites (G∗G and T∗T) is detectable in the range 10.2 ± 10.7 ppm [19]. The sharp signals at 10.85 and 11.85 ppm can be attributed to mismatched G∗T base pairs. [20]. Based on the above-discussed reports, the 1D NMR spectrum of Arab2 was assigned as shown in Fig. 6. For better resolution, the imino region of the 1D proton spectra was acquired from 10 to 13.5 ppm scale. The peaks with chemical shifts at 11.6 and 11.4 ppm correspond to G∗T wobble base pairs [20]. The resonances of imino protons at 11.0 ppm are assigned to G∗G mispair, and at 10.6 ppm are attributed to T∗T mispair [19].

Fig. 6.

One-dimensional 500 MHz 1H NMR spectrum (10–13.5 ppm region) showing the exchangeable (hydrogen-bonded imino) protons of Arab2(T₃AG₃)₂ at 20 °C, in 90% H₂O + 10% D₂O solution containing 20 mM sodium cacodylate (pH 7.4) and 0.1 M NaCl, and 1 M NaCl.

Initially, we contemplated the same intramolecular triplex formed by Arab2 in the presence of 0.1 M Na⁺, as we obtained in the case of Chlm [15] stabilised by G∗G and G∗T bonding in addition to A•T. However, herein, the NMR spectra depicted a slight difference in the bonding patterns, as the NMR peak at 13 ppm for Watson-Crick-Franklin bonded A•T base pairs was found missing. The number of bases in the loop of the proposed structure (Fig. 7a) varies, as we have three Ts in Arab2, while there were four Ts in Chlm sequence (T₄AG₃)₂. Although both the intramolecular structures are stabilised by G∗G and G∗T bonding, they only differ in an additional A•T bond in the case of reported Chlm2 and T∗T in the case of Arab2. Therefore, with a partial difference in the bonding pattern, we can say that sequences of A.thaliana telomere (Arab2) and C. reinhardtii telomere (Chlm2) form a similar type of intramolecular triplex topologies with just a partial difference in base pair (A•T) and mispair (T∗T) bonding patterns. Thus, based on knowledge from the literature and our experimental results, we propose solution structure models representing an intramolecular triplex and tetrad structure formed by Arab2 at 0.1 M and 1.0 M Na⁺ respectively, comprising G∗G and G∗T and T∗T bonding. (Fig. 7b). Further, to determine the structure more accurately the 2D NOESY NMR spectra of Arab2 at such a high salt concentration couldn't be recorded due to inherent limitation of the instrument.

Fig. 7 (a).

A Plausible model of the (a) non-conventional triple helix comprising G∗T∗T, and G∗G ∗T triplets formed by Arab2 at 0.1 M Na⁺ (b) antiparallel quadruplex comprising T∗G∗G∗T formed by Arab2 at 1 M Na⁺.

Fig. 7(b).

Plausible bonding pattern of the antiparallel quadruplex comprising T∗G∗G∗T formed by Arab2.

Thus, based on the facts available in the literature and the experimental results here, we propose a “plausible model” representing an intramolecular triplex and quadruplex structure formed by Arab2 at 0.1 M and 1.0 M Na^+, respectively, comprising the most possible G∗G and G∗T and T∗T bonding (Fig. 7 (a), Fig. 7(b)).

The present finding of a cation-induced structural switch from an intramolecular non-canonical triplex to a non-conventional tetrad is most possibly stabilised by mispairs (G∗G, G∗T, and T∗T) exhibited by a double repeat of Arabidopsis thaliana telomere (T₃AG₃)₂, which has been engaging and calls for further attention regarding such intramolecular structural transitions. It is important to mention here that the formation of an intramolecular purine motif triplex and the DNA quadruplex containing G quartet under physiological pH and ionic conditions by a sequence from the nuclease-sensitive element of c-MYC promoter has already been reported by Belotserkovskii et al. These structures were found to be the general feature of the sequences containing sufficient guanines (homopurine-homopyrimidine stretches) and stabilised by Hoogsteen base pairing between those guanines. [21]. Noticeably, here the two structures, triplex and G-quadruplex, co-exist at physiological conditions, but in the case of Arab2, there is a switch between an intramolecular non-conventional triplex to a tetrad most possibly stabilised by mispairs (G∗G, G∗T, and T∗T) as a function of Na⁺ ion concentration. Generally, the ion atmosphere, in particular, the salt concentration, strongly affects the structure and stability of DNA, as cations are essential for screening the negative charges on the sugar-phosphate backbone. In addition, as is the case here, preferential ion binding of the cations at the backbone or the quad-motif can influence its stability and structure. Consequently, the characteristic properties of these transient structures formed depend not only on the salt concentration and valence of the ions, but also on the ion type. This could be the reason that the molar concentration of Na⁺ specifically facilitated the switching. The change in hydration pattern due to increased cationic concentrations has a large influence on the folding and stability of DNA/RNA structures. Many functional RNAs would not fold under low salt conditions [[22], [23], [24]].

4. Conclusion

To sum up, our results on the structural status of a double repeat of Arabidopsis thaliana telomere are based on a clear correlation between a combination of gel electrophoresis, circular dichroism, optical melting studies, and NMR. Herein, we propose that the double repeat of Arabidopsis thaliana telomere (T₃AG₃)₂ (Arab2) adopts an intramolecular non-conventional triplex in a cation-specific mode, in 0.1 M Na⁺. However, increasing the Na⁺ concentration to 1.0 M facilitates folding, switching to an intramolecular antiparallel tetrad stabilised by the most possible bonding patterns comprising G∗G, G∗T, and T∗T non-Watson-Crick-Franklin bonding. This unique finding of both the non-canonical structural species at low (intramolecular triplex) and high salt (intramolecular antiparallel tetrad) concentrations opens up the possibility of existing unusual tetrads using four nucleobases other than guanines. To the best of our knowledge, such a cation-specific structural transition as a function of salt concentration has not been proposed to date, in a telomeric sequence.

Though we endorse that observation of just exchangeable imino protons in 1D NMR spectra is NOT sufficient to propose a structural model. Even we don't wish to claim that. The conclusion drawn in this report is a result of an agreeable correlation among the techniques used here, giving compelling evidence that the best of non-canonical mispairs (G∗T, G∗G, T∗T) represent non-Watson-Crick bonding, facilitating the formation of the proposed structure. The future studies for high-resolution 2D NMR studies for the unambiguous assignment of the proposed unimolecular triplex and tetrads would require a combination of well-established methods, including MD simulations, enabling a straightforward validation of the proposed plausible model.

Though our studies cannot define the exact in vivo picture and the reported intramolecular transition in the Arab2 telomeric site, they give substantial information about the structural transition of the genomic DNA oligonucleotide in solution with it's possible biological status. There is always a scope for further studies to explore more and more details to establish a link between such structural topologies and their biological relevance.

CRediT authorship contribution statement

Aparna Bansal: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Priyanka Phogat: Writing – review & editing, Data curation. Shrikant Kukreti: Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.