Stability Factors of the Parallel Quadruplexes: DNA vs RNA - The

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Stability Factors of the Parallel Quadruplexes: DNA vs RNA Besik Kankia J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11559 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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The Journal of Physical Chemistry

Stability Factors of the Parallel Quadruplexes: DNA vs RNA

Besik Kankia1,2,*

1Department

2Institute

of Chemistry and Biochemistry, The Ohio State University, Columbus OH 43210, USA

of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia

*Corresponding author: E-mail: [email protected], Telephone: 614-688-8799, Fax: 614-688-5402

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

One of the most stable quadruplexes is formed by G3T sequence (GGGTGGGTGGGTGGG) that folds into a parallel quadruplex with three G-tetrads and chain-reversal T-loops. For example, in 1 mM K+ it unfolds at 75 °C and at physiological conditions it unfolds above 100 °C. The RNA analog, ggguggguggguggg (g3u), which employs exactly same folding topology, demonstrates even higher thermal stability. Here, we performed melting experiments of G3T, g3u and more than 30 chimeric constructs (G3T with RNA nucleotides at certain positions). While g3u quadruplex is 13 °C more stable than G3T, majority of Gg (DNA-for-RNA) substitutions destabilize G3T. Only three Gg and loop Tu substitutions stabilize the structure. However, stabilization effects of these six substitutions overcome destabilization of other nine Gg resulting in higher stability of all-RNA g3u. The present work clearly indicates that the stacking interactions are more favorable in parallel DNA quadruplexes, while the chain-reversal loops play important role in higher stability of RNA quadruplexes. In addition, we have shown that 5'-end of RNA quadruplexes represents more favorable target for stacking interactions than the 3'-end. Based on the current study, rational design of the quadruplexes for particular biotechnological applications and drugs, targeting the quadruplexes, may be envisaged.


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INTRODUCTION Nucleic acid quadruplexes, both DNA and RNA, induce strong interest due to their role in the regulation of gene expression 9.

1-4,

widespread occurrence in aptamers

The latter includes DNA nanowires

endergonic reactions

21

10, 11,

5-8

nanoswitches

and high-affinity DNA couplers

22.

and potential in biotechnological applications 12-17,

detection probes

18-20

driving forces of

The main structural element of quadruplexes is

the guanine (G)-quartet, which is formed by four G-nucleotides (nts) associated through Hoogsteen hydrogen bonds and stabilized by coordination with centrally located cations, such as Na+ or K+. Due to the cation coordination, hydrogen bonds and stacking interactions, the quadruplexes are characterized by high stability. This is particularly true for the GGGTGGGTGGGTGGG (G3T) in the presence of K+, which forms all-parallel quadruplex with chain-reversal single T-loops (Figure 1) and demonstrates unprecedented thermal stability 23, 24. G3T is useful in biotechnologies as a high-affinity coupler 22, building material for DNA nanotechnology 15, 25 and a driving force for endergonic reactions 21. Many aspects of G3T thermodynamics and structural properties have been studied in detail, but its RNA analog, ggguggguggguggg (g3u), has been largely ignored 18, 23, 24, 26-29. In general, studies on different DNA and RNA quadruplexes revealed that RNA analogs are thermodynamically and kinetically more favorable and form predominantly parallel structures, while DNA quadruplexes can adopt both parallel and antiparallel topologies 30-32. The structural homogeneity of RNA quadruplexes is attributed to the constrains in adopting syn glycosidic bonds due to extra hydroxyl groups at 2' positions (2'-OH), that is required for antiparallel topology. Direct comparison of G3T and g3u revealed that in the presence of Na+ both quadruplexes are folded into all-parallel topology and g3u demonstrates 11 °C higher thermal stability 26. To understand reasons for higher stability of RNA quadruplexes, here, we performed comparative thermodynamic study of G3T and g3u quadruplexes, and chimeric constructs containing both DNA and RNA nts in the presence of K+. While all-RNA sequence is ~13 °C more stable than its DNA analog, majority of Gg (DNA-for-RNA) substitutions destabilize the quadruplex; only three Gg substitutions in the top (3'-end) tetrad and loop Tu substitutions increase stability of the structure. The g3u quadruplex is characterized by additional stabilizing interaction between certain g-nts that is absent in the DNA analog. Taken together, the current work demonstrates that the stacking interactions are more favorable in DNA quadruplexes, while the chain-reversal loops play important role in higher stability of RNA quadruplexes.



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MATERIALS AND METHODS All DNA oligonucleotides were obtained from Integrated DNA Technologies. The concentration of the DNA oligonucleotides has determined by measuring UV absorption at 260 nm as described earlier

33.

All

measurements were performed in 0.1 mM KCl, 10 mM Tris-HCl at pH 8.7. UV unfolding/folding experiments were recorded at 295 nm as a function of temperature using a Varian UV–visible spectrophotometer (Cary 100 Bio). Fluorescence measurements of 2-aminopurine (2AP) (ex 310 nm, em 370 nm) were performed using a Varian spectrophotometer (Cary Eclipse). CD spectra were obtained with a Jasco-815 spectropolarimeter. The devices were equipped with thermoelectricallycontrolled cuvette holders. In a typical experiment, oligonucleotide stock solutions were mixed into the desired buffers in optical cuvettes. The solutions were incubated at 95 °C for a few minutes and annealed at room temperature for 10-15 min prior to ramping to the desired starting temperatures. The melting experiments were performed at heating rate of 1 °C/min. All melting experiments of G3T, g3u, chimeric molecules and 2AP-containing constructs revealed cooperative two-state transitions with melting temperature, Tm (±0.5 °C), corresponding to midpoint (50% unfolded quadruplexes) of the transition. The measured dTm values correspond to effects of DNA-for-RNA (DNARNA) substitutions and correspond to stability of the chimeric constructs relative to DNA analog, G3T. CD and UV experiments were conducted at 4 µM concentration of the G3T and g3u domains and the fluorescence measurements were conducted at 2 µM. The Tm values and CD profiles represent average of at least three measurements. The melting curves allowed an estimate of melting temperature, Tm, the midpoint temperature of the unfolding process. Van’t Hoff enthalpies, HvH, were also calculated using the following equations: HvH = 4 R Tm2 / T; R is the gas constant and / T is the slope of the normalized optical absorbance versus temperature curve at the Tm. The Gibbs free energy, at 37 °C, was estimated according to the equation G = HvH (Tm - T) / Tm.

RESULTS AND DISCUSSION


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Before discussing the results, we would like to emphasize that (i) the data in Tables 1-3 and Figures 3-5 correspond to UV melting experiments, while Tables 4 and Figure 6 describe fluorescence melting studies; (ii) all stability effects, dTm, correspond to DNA-for-RNA (DNARNA) substitutions and represent stability effects of chimeric sequences relative to G3T (i.e, dTm of G1g1 substitution is the difference between Tm values of construct #2 and #1, see Table 1); in the fluorescence measurements, the dTm values represent stability effects of chimeric sequences relative to 2AP-G3T or construct #38 (see Table 4); (iii) all constructs demonstrate the same CD profiles (Figure 2) corresponding to all-parallel topology with chain-reversal Tor u-loops suggesting the same tertiary structure. The CD data supported by NMR and X-ray studies demonstrating the same structure for DNA and RNA parallel quadruplexes with anti glycosidic conformations of all guanines

34-36.

CD spectroscopy can't unambiguously determine structure of the

quadruplexes, however, it is a useful tool to characterize overall topology of the quadruplexes and to see whether parallel fold of G3T changes upon DNARNA substitutions. Thus, measured dTm values can be attributed to the local effects of the extra hydroxyl groups, 2'-OH, such as: (i) structural rigidity of RNA vs DNA that constrains glycosidic bonds in anti-conformation and favors parallel quadruplexes

37;

(ii) the C2'

groups of the sugars are positioned such that they are pointing towards the 3'-end 34, 35. The addition of 2'OH creates the steric hindrance between the tetrad and the adjacent tetrad at the 3'-end, which can perturb stacking between these tetrads resulting in destabilization of the quadruplex; (iii) additional interactions (i.e., hydrogen bonds, cation interaction, hydration or hydrophobic interactions) in g3u, which can be observed by comparing dTm values of entire segment substitution with the arithmetic sum of dTm values of corresponding single-nucleotides substitutions dTsum (for details see last paragraph of the following section).

Single-nucleotide substitutions (Gg and Tu) Despite ~13 °C difference in stability of G3T and g3u (construct #1 and #17 in Tables 1 and 2), their CD profiles, as mentioned above, are almost superimposable (Figure 2) and correspond to all-parallel quadruplex topology with chain-reversal T- or u-loops. To figure out which positions are responsible for such a large difference in the thermal stability, we performed systematic study of G3T variants with singlent DNARNA substitutions in all 15 positions (see Table 1). As mentioned earlier, all chimeric constructs



revealed the same CD profiles suggesting that none of the substitutions affect the all-parallel quadruplex topology of G3T (Figure 2). Eight Gg substitutions (in positions 1, 2, 5, 6, 9, 10, 13 and 14) have a destabilization effect (Table 1), G15g doesn't have a measurable effect and the substitutions in remaining six positions (3, 4, 7, 8, 11 and 12) demonstrate stabilization. While compiling the dTm values on the unwrapped 2D map of G3T (Figure 3), the following features were noted: (i) all three loop substitutions (Tu) are accompanied by the same stabilization effects, ~1.3 °C; (ii) Gg substitutions in the bottom Gtetrad (positions 1, 5, 9 and 13) are accompanied by ~0.4 °C destabilization per substitution; (iii) Gg substitutions in the middle G-tetrad (positions 2, 6, 10 and 14) are accompanied by stronger destabilization, ~1.4 °C per substitution; (iv) the substitutions in the top G-tetrad demonstrate strong stabilization effects (~3.6 °C) in positions 3, 7 and 11, however the terminal substitution at the 3'-end, position 15, is not accompanied by any measurable effect. Thus, dTm values of the single-nt substitutions strongly depend on the nucleotide position and varies between -1,4 °C to +4 °C. It is also evident that the substitutions within the same structural motives (i.e., loops or a particular G-tetrad) reveal similar dTm values. Only exception is the top G-tetrad, demonstrating ~3.6 °C stabilization effect for three positions and no impact in the terminal, 15th, position. Interestingly, arithmetical sum of dTm values, of all substitutions, dTsum = 7.6 °C, is significantly smaller than Tm-difference between G3T (construct #1, Table 1) and g3u (construct #17, Table 2), 12.7 °C. Thus, the higher stability of the RNA quadruplex isn't just a sum of individual DNARNA substitutions, indicating on some synergistic interactions within g3u. In other words, non-additive nature of the substitutions indicates on some specific interactions within g3u, which is absent in G3T. In order to find out which nucleotides are involved in these synergistic interactions, dTm and dTsum values of separate structural motives (G-tetrads, loops, GGG-segments) were analyzed in the next sections.

Simultaneous substitutions Simultaneous Tu substitutions. Construct #18 has simultaneous three Tu substitutions and obeys the additivity principle. Specifically, its dTm value, 4 °C, equals to the sum of single Tu substitutions (constructs #5, #9 and #13, Tables 1 and 2). This suggests no interaction between the loops and is in good


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agreement with the structural studies demonstrating that the single-nt loops are completely exposed to solvent without any interaction with the tetrads 34, 35. Single G-tetradg-tetrad substitutions. Due to the zig-zag topology of G3T, (Figure 1), each Gtetrad is formed by four G-nts separated from each other by three positions. For instance, the bottom, or the 5'-end G-tetrad, is formed by G1, G5, G9 and G13. Simultaneous Gg substitutions in these positions result in whole tetrad substitution, or G-tetradg-tetrad. Interestingly, all three G-tetradg-tetrad substitutions reveal different effects; the bottom and middle substitutions (#19 and #20 constructs in Table 2) result in 1.3 °C and 4.5 °C destabilization effects and the top substitution (construct #21) demonstrates 11.7 °C stabilization effect. All three G-tetrad substitutions obey the additivity principle within the experimental uncertainty of Tm. For instance, dTm value of the top G-tetrad substitution (construct #21, Table 2), 11.7 °C, is equal to sum of four Gg substitutions in positions 3, 7, 11 and 15 (3.8+3.7+3.3+0.2=11.0 °C) (Table 1). The additivity suggests that there are no additional interactions between g-nts of the same tetrads and this is true for all three tetrads. The question then arises: why the bottom and middle g-tetrads destabilize the quadruplex, while the top g-tetrad strongly stabilizes it? This can be explained by the steric hindrance of 2'-OH that destabilizes stacking interaction between the tetrad and its neighbor at the 3'-end. Since bottom tetrad (G1-G5-G9-G13, see Figure 1B) and middle tetrad (G2G6-G10-G14) have stacking partners at the 3'-end, they demonstrate destabilization effects. However, top g-tetrad (G3-G7-G11-G15), which is free from the 3'-end stacking doesn't destabilize the quadruplex. In contrast, it shows stabilization effect that should be due to the structural rigidity of RNA or/and stabilizing effect the chain-reversal loops. Simultaneous G-tetradg-tetrad substitutions. The previous sections demonstrated that synergistic interactions in g3u is not caused by interaction of the RNA nucleotides within the same structural motives (i.e., g-nts in the same tetrad or u-loops). It is obvious that stacking interactions between the tetrads should be responsible for the synergy. Here we tested chimeric molecules with simultaneous substitutions of more than one tetrad. Two constructs contain two simultaneous G-tetradg-tetrad substitutions at different positions (constructs #22 and #23, Figure 4). Interestingly, only construct #23 demonstrates synergy: the experimentally measured effect of simultaneous G-tetradg-tetrad in the middle and top positions is exactly twice larger, dTm = 11.0 °C, than the arithmetical sum of each Gg, dTsum = 5.5 °C.



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This clearly indicates that the stacking between the middle and the top g-tetrads is responsible for the synergy. As expected, the synergy is clearly seen when the tetrads are substituted in all three positions (construct #24, Table 2). To figure out if the synergy is maintained at each g-track or ggg, we tested GGGggg substitutions (constructs #25-28). The substitution at the 3'-end (construct #28) revealed 2 °C destabilization, while other positions demonstrated  2 °C stabilization effects. Interestingly, dTm value of the construct #28 is equal to the estimated dTsum value (sum of three Gg substitutions in positions 13, 14 and 15), suggesting no specific interaction between g-nts in these positions. In contrast, remaining GGGggg substitutions (constructs #25-27) demonstrate 1 °C synergy per substitution. Simultaneous two and three GGGggg substitutions (#29 and #30) demonstrate increased stabilization and synergistic effects, while adding the fourth GGGggg substitution at the 3'-end (construct #24), even destabilizes the construct #30. Taken together, the synergy occurs when adjacent guanines are simultaneously substituted in positions 2 and 3, 6 and 7, or 10 and 11. In other words, there are specific interactions between adjacent g-nts (g2-g3, g6-g7 and g10-g11) in the RNA quadruplex, which is absent in the DNA analog. Interestingly, the synergetic effects are associated with the g-nts (g3, g7 and g11) that are adjacent to loop uridines (u4, u8 and u12) and are involved in the loop formation (see Figure 1). Since only these positions (3, 4, 7, 8, 11 and 12) demonstrate stabilization effects, one can conclude that the chain-reversal loops are playing predominant role in the higher stability of RNA quadruplexes.

Stacking between G3T and g3u domains One of the most important observations of this work is that the stacking interaction between DNA tetrads are significantly stronger than between RNA analogs probably due to the steric hindrance of 2'-OH. This can be further tested by melting experiments of two quadruplex domains directly conjugated to each other, (G3T)2 and (g3u)2 (Table 3). It is known that the conjugation radically changes the melting behavior of G3T domain

15, 25.

Specifically, G3T domain demonstrates equilibrium transition at 55 C (Table 1), while the

dimer, (G3T)2, reveals hysteretic loop with unfolding at 85 C and refolding at 55 C (Figure 5)

15.

The

hysteresis loop is attributed to domain assembly and strong stacking interaction between the domains that turns (G3T)2 into an uninterrupted tetrahelix with six G-tetrads (Figures 1D) 15. We would like to emphasize that the stacking between G3T domains involves 3rd and 4th interface tetrads, which are connected to each


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other via single covalent bond, G15-G16. In contrast, all other stacking partners, or tetrads, are connected to each other via four covalent bonds (i.e., 1st and 2nd tetrads are connected via G1-G2, G5-G6, G9-G10 and G13-G14) (Figure 1B). As a result, unstacking of G3T domains from each other is a straightforward process (Figures 1E), while unstacking of the tetrads within G3T domains is topologically constrained. In the case of weak or no stacking between G3T domains, (G3T)2 would melt as two individual G3T domains at ~55 C with plausible pre-melting at lower temperatures due to the domain-domain unstacking. However, (G3T)2 melts as a single cooperative entity at significantly higher temperature, 85 C (Figure 5). This clearly demonstrates that stacking interaction between G3T units is, at least, as strong as stacking within G3T, which turns (G3T)2 into an extra-stable uninterrupted single unit. Interestingly, when stacking between G3T units is intentionally weakened by inserting of T-linkers, the molecule unfolds as two individual G3T units 15, 25.

The unfolding profile of (G3T)2 demonstrates a small pre-melting around Tm of G3T domain, which

might suggest that a fraction of the (G3T)2 unstacks and melts rapidly at this temperature. This pre-melt can also be attributed to failure sequences due to imperfect coupling efficiency of chemical synthesis

15.

More specifically, because G3T quadruplex formation can be inhibited by even a single-nucleotide deletion 24,

truncated (G3T)2 sequences will form one G3T quadruplex with a flapping tail instead of the desired

adjacent two G3T quadruplexes. For instance, a (G3T)2 sequence missing the 5'-terminal guanine would form a single G3T quadruplex with a GGTGGGTGGGTGGG tail at the 5'-end and melt as slightly destabilized G3T domain 15. Thus, dimeric systems, such a s (G3T)2 and (g3u)2 can be employed to test our hypothesis about weaker stacking interactions between g-tetrads. The melting experiments of (g3u)2 reveals equilibrium transition (no hysteresis) with Tm at 70 C (Tables 3) that is only 2 C higher than Tm of g3u (Tables 2), and clearly indicates that stacking interaction between g3u domains is not that strong to produce the extrastable uninterrupted tetrahelix. Interestingly, since (g3u)2 does not demonstrate stacking interaction between g3u domains, both failed and correct sequences unfold around Tm of g3u and therefore premelting, detected for (G3T)2, is not observed (Figure 5). To make sure that formation of the extra-stable tetrahelix is inhibited solely by the interface tetrads (3rd and 4th), (G3T)2 with RNA-RNA interface was tested (construct #35 in Table 3). This chimeric construct also shows equilibrium transition at even lower temperatures, 62 C, and supports our hypothesis about weaker stacking between g-tetrads. Since the



weaker stacking is associated to the g-tetrad at the 5'-end, no hysteresis for RNA-DNA interface (construct # 36) and the large hysteresis for DNA-RNA interface (construct #37) should be observed. In full agreement with the hypothesis, RNA-DNA interface shows equilibrium transition, and DNA-RNA interface regains hysteretic behavior characteristic to the strong stacking interaction (Figure 5). Additional evidence that only DNA-DNA (construct #33) and DNA-RNA (construct #37) interfaces are suitable for the strong stacking between the domains comes from CD measurements (Figure 2C): only these constructs demonstrate higher CD amplitudes due to the extra stacking interactions 25. Thus, stacking between RNA tetrads are significantly weaker, however, strong stacking interaction is possible between parallel DNA and RNA quadruplex when interaction occurs at the 5'-end of the RNA quadruplex.

Tailoring quadruplex sequence to modify stability It was demonstrated that the free energy of G3T quadruplexes can be used to drive unfavorable (endergonic) reactions of nucleic acids

21.

In general, G3T sequences can be incorporated within DNA

duplexes, which after interaction with an initiator (i.e., pathogen DNA in non-enzymatic signal amplification or DNA polymerase in PCR) self-dissociate from the complementary strand and fold into quadruplexes. The key point of quadruplex-driven reactions is that the quadruplexes form with significantly more favorable thermodynamics than the corresponding DNA duplexes

21.

The self-dissociation energy comes from the

difference between free energies of the quadruplex and the corresponding duplex. For instance, isothermal nature of quadruplex priming amplification (QPA), is based on the fact that, in the presence of K+ ions, 15bp long G3T duplex (G3T with the complement) demonstrates 5 °C less stability than its truncated 13-bp version (missing two terminal Gs)

21.

Since the truncated version of G3T is unable to form quadruplex, its

corresponding duplex demonstrates typical equilibrium transition at predicted, by nearest-neighbor analysis, temperature. However, unfolding of G3T duplex is a nonequilibrium process due to quadruplex formation of the released strands that significantly destabilizes the duplex and shifts the transition to lower temperatures. As a result, shorter duplex is approximately 5 °C more stable than the longer one. Thus, around Tm of the shorter duplex, ~50% of the truncated G3T can bind to target (or complement) and serve as a primer. After adding missing Gs by polymerase, the duplex destabilizes by ~5 °C that facilitates its


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dissociation and the next priming round without the need for thermal denaturation. The rate of QPA is limited by the temperature difference between these duplexes 21. Specifically, by performing amplification at higher temperatures we facilitate self-dissociation, however, impede the priming process. The QPA system with larger temperature difference between the primer and product can facilitate both priming and selfdissociation and, as a result, increase amplification rate. Thus, we hypothesize that designing more stable quadruplexes can facilitate quadruplex-driven reactions. Since the stabilization effects of Gg substitutions strongly depend on the position, one can design quadruplexes with desired stability by tailoring its sequence. For instance, stability of all-RNA g3u can be reached and even exceeded by few DNARNA substitutions. Indeed, the construct #30 with only three Gg substitutions at positions 3, 7 and 11 reveals 11.2 °C increase in G3T stability, and additional three Tu substitutions, construct #32, shows 4.2 °C higher stability than all-RNA g3u (Table 2 and Figure 4). We used van’t Hoff analysis to determine the unfolding enthalpies, HvH, for the quadruplex unfolding (see Materials and Methods). For G3T, HvH = 62±5 kcal/mol, while more stable RNA quadruplex, g3u shows higher endothermic heat, 71±7 kcal/mol. All chimeric quadruplexes revealed values between these two estimations. Free energies, G at 37 °C, are equal to 3.4 kcal/mol (for G3T) and 6.5 kcal/mol (for g3u). Thus, RNA quadruplex is thermodynamically more favorable than its DNA analog.

Fluorescence measurements To confirm the present data with an additional technique, fluorescence spectroscopy of 2AP-modified sequences was employed. 2AP is a fluorescent analog of adenine, forms Watson-Crick base pairs with thymidine 38, 39 and has a high quantum yield (0.68) as a free base 40. G3T quadruplex tolerates nucleotide substitutions at all loop positions without any changes in the tertiary structure

18, 28.

However, the purine

nucleotides (i.e., A, 2AP, G, 6-methylisoxanthopterin) slightly destabilize the quadruplex due to the stronger stacking of purine bases with adjacent Gs, which has to be overcome during rearrangement of the sequence into four GGG-tracks to form a monomolecular quadruplex (construct #38 in Table 4) 18, 28. G3T with T2AP substitutions at all three loop positions demonstrate a remarkable 100-fold increase in fluorescence upon quadruplex formation and is very useful to study quadruplex stability (prior to quadruplex formation the fluorescence is efficiently quenched by surrounding Gs, while in the quadruplex 2AP is fully



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accessible to solvent and regains the fluorescence) 18, 21). The melting experiments of 2AP-G3T monitored by both UV and fluorescence signal revealed identical Tm values

21.

Thus, while UV-absorption and 2AP-

fluorescence are sensitive to different optical properties of the G3T quadruplex, both accurately monitor overall unfolding of the tertiary structure. We performed fluorescence melting experiments of 2AP-G3T, 2AP-g3u and 2AP-modified several key chimeric molecules (see Table 4 and Figure 6). Specifically, we tested all G-tetradg-tetrad substitutions (Constructs #40, #41 and 42) and construct #43 containing only stabilizing substitutions and demonstrating the highest stability. As expected, all fluorescence Tm values are shifted to lower temperatures by  5 C due to 2AP destabilization, however, closely correlate with the UV melting data (Table 4) demonstrating perfect agreement between the methods.

CONCLUSIONS Despite the fact that all-RNA quadruplex is 13 °C more stable than its DNA analog, majority of Gg substitutions destabilize the quadruplex. Only three Gg substitutions in the top (3'-end) tetrad and loop Tu substitutions increase stability of the structure. The single-nucleotide substitutions within the same structural motives (i.e., loops or a particular Gtetrad) reveal similar dTm values. Only exception is the top G-tetrad, demonstrating ~3.6 °C stabilization effect for three positions and no impact in the terminal, 15th, position. The effect of G-tetradg-tetrad substitution strongly depends on the position; the bottom (at the 5'-end) and middle substitutions destabilize the quadruplex by 1.3 °C and 4.5 °C, while top tetrad (at the 3'end) demonstrates 11.7 °C stabilization effect. The g3u quadruplex is characterized by additional stabilizing interaction between certain g-nts that is absent in the DNA analog. While stacking interactions are more favorable in G3T quadruplex, the chainreversal loops play important role in higher stability of g3u.

ACKNOWLEDGMENTS This work was funded by the Shota Rustaveli National Science Foundation (Grant FR17_140).


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FIGURE CAPTIONS

Figure 1. 3D representation of G3T quadruplex with all parallel GGG-segments (spheres connected with black lines) and chain-reversal T-loops (blue bars) (A); surface of 3D model of G3T unwrapped into 2D map (B); simplified schematic of G3T domain (C); 3D representation of G3T dimer, (G3T)2 (D); simplified schematic of (G3T)2 demonstrating unstacking of G3T domains (E). Gray discs and stripes represent Gquartets.

Figure 2. CD profiles of G3T (blue) and g3u (red) (A), all chimeric domains (B) and dimeric quadruplexes (C). Buffer: 0.1 mM KCl, 10 mM Tris-HCl at pH 8.7.

Figure 3. 2D map of G3T with stability effects, dTm, of single-nucleotide Gg and Tu substitutions.

Figure 4. Bar graphs of stability effects, dTm, of constructs with multiple substitutions. The horizontal blue and red lines correspond to the stability levels of al-DNA G3T and all-RNA g3u.

Figure 5. UV unfolding (solid) and refolding (dashed) curves of (G3T)2, (g3u)2 and chimeric dimers in 0.1 mM KCl. Curves are offset for clarity. For schematics and the corresponding sequences see Table 3.

Figure 6. Representative fluorescence unfolding curves of 2AP-2ndg4 and 2AP-stable Q(2) in 0.1 mM KCl. For actual sequences see Table 4.


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Table of Contents


Stability Factors of the Parallel Quadruplexes: DNA vs RNA - The

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