RNA Triplex-to-Duplex and Duplex-to-Triplex Conversion According to
a Temperature-Controlled Cycle Induced by Coralyne
Francisco J. Hoyuelos, a Begoña García,* a José M. Leal,a Natalia Busto,a Tarita Biver,b
Fernando Secco* b and Marcella Venturini b
aUniversidad de Burgos, Departamento de Química, 09001 Burgos, Spain.
bUniversità di Pisa, Dipartimento di Chimica e Chimica Industriale, 56126 Pisa, Italy.
Abstract
Spectrophotometric, calorimetric, displacement assay and kinetic analyses of the binding of the fluorescent dye coralyne to poly(A)2poly(U) have served to enlighten the dye ability to bring about dramatic changes in the RNA structure. The sets of data gathered agree to convey that coralyne is able to induce the triplex-to-duplex conversion and also the duplex-to-triplex conversion according to a thermodynamic cycle driven by temperature, provided that the [dye]/[polymer] ratio (CD/CP ) is kept constant at a value
higher than unity. Alternatively, at constant temperature, a cycle driven by the CD/CP ratio
can also be devised.
1. Introduction
Over the last few years, non-canonical nucleic acid structures have aroused a great deal of interest due to their involvement in biological processes.1-4 In particular, triplex
structures have been given paramount importance in therapies based on antigene and antisense strategies.5-7 On the other hand, coralyne (Figure 1) is able to modulate
conformational changes in molecular beacons useful for selective nucleic acids detection8
and to induce disproportionation of double stranded DNA into triplex and single strands.9
Moreover, it has been shown recently that also RNA, in the poly(A)poly(U) form, disproportionates at 25 °C in the presence of coralyne according to reaction (1), provided
that the duplex (AU) is present in excess, i.e., in a [dye]/[polymer] ratio (CD/CP) less than
0.8. 10
(1)
Moreover, melting experiments have shown that, for CD/CP > 0.8, AUD also
disproportionate into triplex and single strands for temperatures above 40 °C. In this case, the process is represented by reaction (2):
(2)
Fig. 1 Coralyne chloride (8-methyl-2,3,10,11-tetramethoxydibenzo[a,g]-quinolizinium chloride).
Therefore, coralyne is able to induce duplex-to-triplex transition whereas, for instance, proflavine or ethidium are not, and this issue needs to be justified. All these drugs bind to poly(A)poly(U) by intercalation11,12 but, in the case of the crescent-shaped
coralyne, a notable portion of the intercalated drug remains partially outside the backbone of the duplex and can react with a second duplex unit by intercalation.10 Under these
circumstances, the encounter of two AUD molecules gives way to an intermediate species that evolves to the UAUD triplex with concomitant formation of AD, according to reaction (2). Surprisingly, the melting curve of poly(A)2poly(U) at CD/CP = 3.0 was very
similar to that of poly(A)poly(U) also at CD/CP = 3.0.10 This striking behaviour has been
explained assuming that the excess of coralyne can further the release of a poly(U) strand from UAUD at room temperature, according to reaction (3), in a way such that the species
AUD + AU UAUD + A
effectively present at 25 °C is the AUD duplex in a poly(A)2poly(U) solution with dye in excess:
(3) It should be noted that the pronounced affinity of coralyne for A has been suggested as the driving force for reaction (2).10 In a similar way, the notable affinity of the dye with
the U strand under CD/CP > 1 conditions (K = (4.6 ± 0.5) × 105 M-1 measured in our
laboratory) might afford the driving force for reaction (3). To reliably assess this behaviour, we have undertaken spectrophotometric, kinetic, calorimetric and ethidium bromide displacement assay studies on the coralyne/poly(A)poly(U) and coralyne/poly(A)2poly(U) systems.
2. Materials and Methods
2.1 Materials. Coralyne chloride (Sigma-Aldrich, 99.9%) was used without further purification. Stock solutions of the dye (ca. 2×10-3 M) were prepared by dissolving
weighed amounts of the solid in water and kept in a refrigerator (4 °C). Polyuridylic acid (poly(U)) and poly(A)poly(U) were lyophilized sodium salts from Sigma-Aldrich. Other reagents were analytical grade. Stock solutions of the polynucleotides were standardized spectrophotometrically (I = 0.1 M (NaCl), pH = 7.0), using = 8900 M-1 cm-1 (260 nm) for
poly(U), and = 14900 M-1 cm-1 (260 nm) for poly(A)poly(U).13 Poly(A)2poly(U) was
obtained by quantitative reaction at pH = 7.0 between equimolar amounts of poly(U) and poly(A)poly(U). The analytical concentration of poly(A)2poly(U), CP, is expressed in
molarity of base triplets, whereas that of coralyne is denoted as CD. The absorbance of the
dye and polynucleotide stock solutions showed no noticeable changes over a month, revealing sufficient stability over such long time. Sodium chloride was used to adjust the ionic strength and sodium cacodylate ((CH3)2AsO2Na, 1.0×10-2 M) was employed to keep
the solutions pH constant at 7.0. Doubly distilled water was used throughout.
2.2 Methods. pH measurements were taken with a Metrohm 713 pH-meter. Spectrophotometric measurements were performed on a Hewlett-Packard 8453A (Agilent Technologies, Palo Alto, CA) photodiode array spectrophotometer fitted out with photodiode array detection and computer-assisted temperature control systems (±0.1ºC); this instrument was also employed to monitor the kinetic experiments, in which the kinetic curves were transferred to a PC and evaluated with the Table Curve fitting program (AISN software, Mapleton, OR).
Ethidium bromide displacement assay experiments were carried out on a Shimadzu Corporation RF-5301PC spectrofluorometer (Duisburg, Germany). Different solutions (coralyne and EB alone, coralyne/duplex and coralyne/triplex at CD/CP = 0.3, 1.0, 1.5 and
13.0 were titrated with increasing amounts of ethidium bromide. The fluorescence spectra were recorded at both coralyne (λexc = 420 nm, λem = 470 nm) and ethidium bromide (λexc =
510 nm, λem = 598 nm) wavelenghts.
Circular dichroism (CD) measurements were obtained with a thermostatted MOS-450 Biologic spectrometer fitted out with a 1.0 cm path-length cell. CD titrations were carried out at 25 °C by adding increasing amounts of the dye to a known volume of the polymer solution.
Differential scanning calorimetry (DSC) experiments were carried out on a Nano DSC Series III system of TA Instruments with a temperature scan rate of 0.2 °C min-1; the
solutions were degasified with a TA Instruments for 30 minutes before use. The thermograms collected were analysed with the NanoAnalyze 2.0 software. The buffer-buffer base line was run by at least five heating/cooling cycles, until the heating was reproducible; then, it was subtracted from the sample data.
Figure 2 shows the melting curves of the poly(A)2poly(U)/coralyne system for CD/CP
ratios from 0.3 up to 3.0. For CD/CP = 0.3, the triplex initially present shows the UAUD
AUD + U transition at 58 °C. Full denaturation to single strands commences near 80 °C. The same melting features are still observable at CD/CP = 1.0; even though the triplex
duplex transition is less evident, the melting profile shows that the UAUD complex initially present releases a U strand, yielding the AUD duplex (Tm = 58 °C). For CD/CP =
1.5, the melting profile dramatically changed; actually, the absorbance remained constant after a temperature increase up to 40 °C and then decreased suddenly, reaching a minimum whose location also depends on the CD/CP ratio. The observed hypochromic effect reveals
a transition accompanied by enhanced helicity, which can be accounted for only by the duplex triplex transition. It can then be concluded that, at CD/CP > 1.0, the triplex
present in the sample converts into the duplex according to reaction (3), and that the stable species at room temperature is the duplex. On the other hand, the hypochromic effect observed between 40 °C and 60 °C is ascribed to formation of the triplex according to reaction (2). The same effect is displayed for CD/CP = 3.0 but, in this case, the duplex
triplex transition shifts to lower temperatures.
The sequence of the melting profiles (Figure 2) shows a stable temperature range for duplex formation according to reaction (3), and becomes shortened as the dye content is raised. Comparison of the data with the analogous plot sequence obtained starting from the duplex,10 confirms the above assumption. Further temperature increase causes the triplex
at the minimum of the melting curve to undergo direct denaturation to single strands, according to reaction (4),
The interpretation of the processes that occur prior to and concurrent with the melting experiments is reinforced by the observation that the amplitude of the triplex duplex transition at CD/CP = 0.3 is (roughly) half the absorbance jump at CD/CP =
3.0 in the first downward stretch of the melting curve assigned to the duplex triplex transition. Actually, according to reaction (2), two duplex units, which prevail at 25 °C, are required to form a single triplex unit in the first branch (CD/CP = 3.0), whereas every
triplex unit formed yields one duplex molecule according to reaction (3) (CD/CP = 0.3).
Fig. 2 Melting profile of the triplex coralyne/poly(A)2poly(U) system at different CD/CP ratios: 0.3, 1.0, 1.5 and 3.0. CD = 1.7 × 10-5 M, [NaCl] = 0.10 M, pH = 7.0, λ = 280
nm.
Figure 3A plots the minimum temperature (Tmin) vs. CD/CP values. A sudden decrease
of the minimum temperature is observed in the 2.0 - 2.5 CD/CP range. The amplitude of the
first branch of the melting curve (Figure 3B) displays symmetrical behaviour. For CD/CP ≥
3.0, the constancy of both Tmin and amplitude indicates that the duplex-to-triplex
conversion is quantitative under such conditions.
40 60 80 0.3 0.4 0.5 0.6 A b s T, ºC 1.5 40 60 80 0.3 0.4 0.5 0.6 A bs T, ºC 3.0 40 60 80 0.4 0.5 0.6 0.7 T, ºC A b s 1.0 40 60 80 1.2 1.4 1.6 1.8 T, ºC A bs 0.3 Duplex Triplex Duplex Triplex
Fig. 3 Variation with CD/CP of (A) minimum temperature (Tmin), and (B) amplitude
of the first branch of the melting curve for coralyne/poly(A)2poly(U), [NaCl] = 0.10 M, pH = 7.0.
Figure 4 shows the kinetic behaviour of a poly(A)2poly(U) and coralyne mixture at 25°C. The increase in absorbance for CD/CP = 3.0, curve (a), reveals that the triplex
evolves to duplex at this temperature. By contrast, absence of any kinetic effect for CD/CP = 0.3, curve (b), reveals that the triplex remains unaltered, in agreement with the
observed stability of UAUD under such conditions.
Fig. 4 Kinetic experiments performed by mixing coralyne (CD = 3.40 × 10-5 M) and
poly(A)2poly(U) for (a) CD/CP = 3.0 and (b) CD/CP = 0.3; pH = 7.0, [NaCl] = 0.1 M , λ =
280 nm, T = 25 °C.
Figure 5 shows the CD spectra of poly(A)2poly(U) at different CD/CP ratios. The CD
spectra, of the triplex recorded in the UV range characteristic of the polymer (Figure 5A), show a decrease in intensity of the 260 nm band at CD/CP < 1; the well-defined isoelliptic
point at 280 nm can be accounted for by the formation of UAUD starting from UAU and D. For CD/CP > 1, the remarkable deviations of the isoelliptic point reveal a profound
100 200 300 400 0.00 0.02 0.04 0.06 a A bs t , s b 1.5 2.0 2.5 3.0 3.5 35 40 45 50 55 60 Tm in , °C CD/CP A 1.5 2.0 2.5 3.0 3.5 1 2 3 4 5 10 -4 A bs /C P , M -1 CD/CP B
change in the structural properties of the system, ascribable to conversion of the triplex into the corresponding duplex according to reaction (3). Figure 5B also shows the somewhat less intense but unequivocal rise of an induced CD signal of the dye in the VIS region.
Fig. 5 Circular dichroism spectra recorded on addition of increasing coralyne amounts to poly(A)2poly(U). CP = 6.2 × 10-5 M, CD/CP = 0 - 2.5, [NaCl] = 0.10 M, pH =
7.0, T = 25 °C. Arrows indicate the change with the dye increase.
Fig. 6 Molar ellipticity [] vs. CD/CP plot for the coralyne/poly(A)2poly(U) system.
CP = 6.2 × 10-5 M, [NaCl] = 0.10 M, pH = 7.0, λ = 340 nm, T = 25 °C.
Figure 6 represents the CD induced behaviour at 340 nm, showing a typical binding isotherm for CD/CP ≤ 1, which corresponds to the reaction UAU + D → UAUD. The
isothermal triplex-to-duplex conversion commences at CD/CP > 1, concurrent with the
250 275 300 325 0 20 40 60 10 -3 , D eg M -1 c m -1 , nm A 350 400 450 0 1 2 3 4 5 B 10 -3 , D eg M -1 c m -1 , nm 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 10 -3 [ ], D eg M -1 c m -1 CD/CP
conclusions drawn from the UV melting (Figure 2) and kinetic (Figure 4) measurements, as well as with the DSC experiments described below.
Figure 7A shows the CD melting curve of the coralyne/poly(A)2poly(U) system at CD/CP = 0.3. Under these conditions, the UAUD triplex predominates at room
temperature and undergoes denaturation at 58 °C, in agreement with the UV melting data (Figure 2) and DSC experiments (see below). At CD/CP = 3.0, the melting curve of same
system (Figure 7B) concurs well with previous observations. The well defined dichroic jump reveals that the transition (observed in the UV melting experiments) occurs at 33 °C. This feature also means that, when the temperature rose to 33 °C, the AUD duplex formed at room temperture disproportionates into triplex and single strand according to reaction (2). The find that the final dichroism (UAUD) was lower than the initial one (AUD) appears to be contradictory as long as [θ]AUD < [θ]UAUD (Figure 7A). This apparent
contradiction, however, can be accounted considering that the formation of one triplex unit according to reaction (2) requires two duplex units.
Fig. 7 CD melting curves for the coralyne/poly(A)2poly(U) system monitored for CD/CP =
0.3 and 3.0. CD = 1.7 × 10-5 M, [NaCl] = 0.10 M, pH = 7.0, λ = 280 nm.
Figure 8 shows the effect of the CD/CP ratio on the DSC thermograms. The peak at
CD/CP = 0 corresponds to full denaturation of poly(A)2poly(U) (Tm = 58 °C). Further
addition of coralyne up to CD/CP = 0.3 results in two overlapped peaks that correspond to 20 30 40 50 60 70 80 0 1 2 3 4 0.3 1 0 -4 , D e g M -1 cm -1 T, °C A 20 30 40 50 60 70 80 -1 0 1 2 3 3.0 1 0 -5 , D e g M -1 c m -1 T, °C B
the triplexduplexsingle strand transitions. For CD/CP = 1.0, the left maximum shifts to
lower temperature, whereas the location of the maximum at 58 °C remains unaltered. Finally, for CD/CP = 3.0 a single broad band appeared, in which the duplex triplex and
triplex single strand transitions overlapped, in agreement with the behaviour of the UV melting experiments. In conclusion, the UV, CD and DSC melting experiments and the kinetic measurements concur with the conclusion that coralyne promotes the triplex-to-duplex conversion at room temperature.
20 40 60 3.0 1.7 1.0 0.3 M ol ar H ea t C ap ac ity , k J/ m ol K T, °C 0
Fig. 8 DSC thermograms for the coralyne/poly(A)2poly(U) system at different CD/CP
ratios; CP = 2.5×10-4 M, CD/CP = 0 to 3.0, [NaCl] = 0.10 M, pH = 7.0, temperature scan
rate = 0.2 °C/min.
Finally, displacement assays were carried out by adding ethidium bromide (EB) to the coralyne/triplex and coralyne/duplex complexes at different CD/CP ratios. In view that
both EB/RNA and the coralyne/RNA complexes are fluorescent, the fluorescence intensity was recorded in each case at the characteristic wavelengths of the two species, (λem = 598
nm, λexc =510 nm) and (λem = 470 nm, λexc = 420 nm) respectively. It must be recalled here
fluoresecence, in both cases as a consequence of intercalation. Figure 9A shows the fluorescence recorded at 598 nm upon addition of EB for the duplex RNA/coralyne and triplex RNA/coralyne, both at CD/CP = 0.3. In agreement with earlier results10 and the
above discussion (Figure 2), the duplex structure was converted into triplex at this coralyne content. Actually, the observed tendency is similar starting from either the duplex or the triplex. In view that the affinity of the coralyne/poly(A)2poly(U) system (Kapp = 2.3
×107 M-1)10 was greater than that of ethidium/poly(A)2poly(U) (K
app= 6.2× 104 M-1)14 it can
be surmised that, at CD/CP = 0.3, the enhanced fluorescence up to the plateau upon addition
of EB is ascribable to formation of the ternary, doubly intercalated, coralyne/EB/ poly(A)2poly(U).
Figure 9B shows the result when the fluorescence of coralyne is measured at 470 nm under conditions identical with those of Figure 9A. A similar tendency was observed starting from either the duplex or the triplex, but the fluorescence intensity starting from the duplex is roughly half that starting from the triplex. This result can be explained with reaction 2, which makes clear that two duplex molecules are required to yield the triplex, that is, the resulting triplex concentration is half starting from the duplex compared with that starting from the triplex. The two curves of Figure 9B show that, at the very beginning, EB diminishes the fluorescence of the coralyne/triplex because the coralyne/EB/triplex ternary complex is formed. The formation constant obtained from the initial range of the Stern Volmer equation is 1.4×105 M-1 (Figure 10A). Afterwards it is
observed a slight increase in fluorescence, yielding a minimum, due to the partial removal of coralyne from the interior of the triplex to give a ternary complex with smaller fraction of intercalated coralyne and, hence, more accessible to the quencher. The final descending stretch comes together because at high quencher concentrations the overall coralyne fraction accessible is similar.
The same experiments were performed at CD/CP = 1 and 1.5, in which the duplex is
present independently of the initial starting material, either the duplex or coralyne/triplex10
and present results (Figure 2). EB/RNA gave similar enhanced fluorescence, only sligthly higher than that of EB alone (Figure 9C), indicating that the coralyne/EB/ poly(A)poly(U) ternary system does not form under these conditions due to the initial saturation of the duplex by coralyne. The difference between the affinity constants of the coralyne/ poly(A)poly(U) (Kapp = 1.8×106 M-1) and EB/poly(A)poly(U) (Kapp = 2.6×104 M-1)
systems15 bears out this interpretation. Figure 9D was plotted under same conditions than
Figure 9C, this time observing the coralyne/RNA emission. Quenching was observed over the whole concentration range. The fluorescence of the system with initial duplex fits well to the Stern Volmer equation (Figure 10B), with Ks=1.3×103 M-1 the dynamic quenching
constant, whereas the quenching is not linear starting from the triplex because, according to reaction 3, several different fluorescent species are at equilibrium. The quenching effect of EB is now larger, denoting that UAUD and UD are quenched stronger than UAD. The experiments were repeated at CD/CP = 3.0; however, as a consequence of the large excess
of coralyne outside the helix, only the quenching with EB was observable.
The set of thermodynamic and kinetic results are consistent with the presence of poly(A)2poly(U) at low coralyne concentration and with the poly(A)poly(U) at high coralyne concentration.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 10 20 30 40 50 60 F59 8 nm 104 ·C EB A 0.0 0.2 0.4 0.6 0.8 1.0 1.2 40 80 120 F470 n m 104 ·C EB B 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 10 20 30 40 50 F59 8 nm 104 ·CEB C 0.0 0.2 0.4 0.6 0.8 1.0 1.2 40 80 120 F470 n m 104 ·C EB D
Fig. 9 Fluorescence intensity as function of EB concentration. A) from coralyne/ poly(A)2poly(U), CD/CP = 0.3 (▼)and from coralyne/poly(A)poly(U) CD/CP = 0.3 (■) λexc
= 510 nm, λem = 598 nm. B). Similar data of A, λexc = 420 nm, λem = 470 nm, C) From
coralyne/poly(A)2poly(U), CD/CP = 1.5 (
♦
),
from coralyne/poly(A)poly(U), CD/CP = 1.0(●), and (C) ethidium bromide alone (▲), λexc = 510 nm, λem = 598 nm. (D) Similar data of
A corrected by the dilution factor, λexc = 420 nm, λem = 470 nm.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.9 1.0 1.1 1.2 1.3 1.4 1.5 F0 /F 106 ·CEB A 0 1 2 3 4 5 6 1.0 1.2 1.4 1.6 1.8 F0 /F 105 ·C EB B
Fig. 10 Stern-Volmer plots of the coralyne/poly(A)2poly(U), CD/CP = 0.3 (A) and coralyne/
The results of the present work can be combined with those reported for the disproportionation coralyne induced duplex triplex + single strand.10 It turns out that the
reactions involved in the two processes, reactions (2) and (3), could constitute a cycle operated by temperature changes (Figure 11). The set of results show that, under excess of dye, the coralyne/RNA system undergoes a cycle controlled by two key temperatures (T2 >
T1). At T2, the duplex disproportionates into triplex, whereas at T1 (T2 > T1) the triplex
converts into duplex. A cycle operated by a change of the CD/CP ratio can also be devised:
for CD/CP < 1, the duplex disproportionates into triplex, whereas, for CD/CP > 1, the triplex
converts into duplex. It should be noted that, in both cycles, two duplex units are consumed for every duplex reformed, in such a way that in only few cycles the fraction of reacting species would be rapidly reduced, unless sufficient time is given to allow regeneration of AU by reaction between the A and U strands produced in reactions (2) and (3).
Fig. 11 Reactions (2) and (3) combine to give the cyclic process prompted by
temperature changes. For CD/CP > 1.0, one UAUD unit yields one AUD unit, (reaction
(3)), at the lowest temperature, T1. On the other hand, two units of the formed duplex yield
one triplex unit, (reaction (2)), at the highest temperature T2.
4. Conclusion
The results obtained show that the duplex and the triplex complexes inter-convert one into the other. This process, however, comes about not according to a reversible reaction in which the direct and reverse processes go the same pathway (same activated complex), but rather through the thermodynamic cycle shown in Figure 11. This cyclic inter-conversion is promoted by coralyne.
Acknowledgments
The financial support by Ministerio de Educación y Ciencia, Project CTQ2009-13051/BQU, supported by FEDER, Junta de Castilla y León, Project BU-299A12-1 and Obra Social “la Caixa”, Spain, are gratefully acknowledged.
References
1 D.-L. Ma, D. S.-H. Chan, P. Lee, M. H.-T. Kwan, C.-H. Leung, Biochimie 2011, 93, 1252-1266
2 C. Sissi, B. Gatto, M. Palumbo, Biochimie 2011, 93, 1219-1230
3 P. Catasti, X. Chen, S. V. S. Mariappan, E. M. Bradbury, G. Gupta, Genetica 1999, 106, 15-36
4 S. V. S. Mariappan, P. Catasti, L. A. Silks, E. M. Bradbury, G. Gupta, J. Mol. Biol. 1999, 285, 2035-2052.
5 A. K. Field, Curr. Opin. Mol. Ther. 1999, 1, 323-331
6 M. L. Jain, P. Y. Bruice, I. E. Szabo, T. C. Bruice, Chem. Rev. 2012, 112, 1284-1309
7 A. K. Jain, S. Bhattacharya, Bioconjugate Chem. 2010, 21, 1389-1403.
8 Y.-H. Lin, W.-L. Tseng, Chem. Commun., 2012, 48, 6262–6264
9 M. Polak, N. V. Hud, Nucleic Acids Res. 2002, 30, 983-992.
10 T. Biver, A. Boggioni, B. Garcia, J. M. Leal, R. Ruiz, F. Secco, M. Venturini, Nucleic Acids Res. 2010, 38, 1697-1710.
11 T. Biver, F. Secco, M. Venturini, Arch. Biochem. Biophys.2005, 437, 215-223 12 Y. Babayan, G. Manzini, L. E. Xodo, F. Quadrifoglio, Nucleic Acids Res. 1987, 15,
5803-5812.
13 B. Janik, in Physicochemical Characteristics of Oligonucleotides and Polynucleotides, IFI Plenum, New York-Washington-London, 1971.
14 B. Garcia, J. M. Leal, V. Paiotta, S. Ibeas, R. Ruiz, F. Secco, M. Venturini, J. Phys. Chem. B 2006, 110, 16131-16138.
15 M. L. D'Amico, V. Paiotta, F. Secco, M. Venturini, J. Phys. Chem. B 2002, 106, 12635-12641.
