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A mirror-image tetramolecular DNA quadruplexw Phong Lan Thao Tran,ab Rui Moriyama,c Atsushi Maruyama,c Bernard Raynerab and Jean-Louis Mergny*ab Received 4th March 2011, Accepted 25th March 2011 DOI: 10.1039/c1cc11293g L-DNA, the mirror image of natural DNA forms structures of opposite chirality. We demonstrate here that a short guanine rich L-DNA strand forms a tetramolecular quadruplex with the same properties as a D-DNA strand of identical sequence, besides an inverted circular dichroism spectra. L- and D-strands self exclude when mixed together, showing that the controlled parallel self-assembly of different G-rich strands can be obtained through L-DNA use. L-Nucleic acids, the mirror images of natural D-DNA and RNA, have found a number of interesting applications in biotechnology (as aptamers,1,2 PCR3 or microarray4 probes, agents for enantiomeric separation5 and molecular beacons)6 and nanotechnology.7 One of the advantages of L-nucleic acids is their nuclease resistance, allowing one spiegelmer (from ‘‘Spiegel’’ meaning mirror in German) to enter clinical trials.8 As a perfect mirror image of D-DNA, L-DNA forms duplexes with identical physical characteristics (solubility, stability) except for chirality, leading to left-handed double-helices.9 We investigate here their potential to form G-quadruplexes,10 using the well known tetramolecular [TG4T]4 complex as a model.11 2 0 -Deoxy-L-guanosine and L-thymidine were prepared according to slightly modified procedures.12,13 N-2-Isobutyryl-20 deoxy-L-guanosine and L-thymidine were dimethoxytritylated and further converted to the corresponding b-cyanoethylphosphoramidites according to standard procedures. L-Oligonucleotides were assembled on a DNA synthesizer, deprotected and purified by reverse-phase HPLC (see full details in ESIw). We expected that a short L-TG4T strand should form a stable tetramolecular quadruplex with identical properties as its well-known regular DNA counterpart but opposite CD spectra. Fig. 1 summarizes the evidence for G4 formation obtained for L-TG4T. L- and D-strands form complexes of identical retarded mobility when incubated under conditions favoring G4 formation. Isothermal difference spectra (IDS) were obtained by calculating the difference between the absorbance spectra of the folded and unfolded forms of a sample
a
Universite´ de Bordeaux, Laboratoire ARNA, F-33000 Bordeaux, France. E-mail:
[email protected] b INSERM, U869, Laboratoire ARNA, IECB, F-33600 Pessac, France c Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan w Electronic supplementary information (ESI) available: Experimental procedures, CCC formulae, representative kinetic and gel experiments. See DOI: 10.1039/c1cc11293g
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The Royal Society of Chemistry 2011
Fig. 1 Evidence for G4 formation by L- and D-TG4T. Left: IDS spectra of the D- and L-strands in 0.5 M KCl. Gel: migration of D- and L-strands in 20% polyacrylamide under conditions favorable (+) or not () to G4 formation (60 mM strand concentration). ‘‘M’’ lanes correspond to oligo dTn migration markers. Right: CD spectra of the L- and D- quadruplexes in 0.5 M KCl.
(after and before an isothermal kinetics experiment);14 they are nearly superimposable (left) while circular dichroism (CD) spectra are inverted (right), as expected for a left-handed L-DNA quadruplex. We then compared the thermal stability and kinetics of the two quadruplexes. First, the apparent melting temperatures11 of the preformed L- and D-DNA quadruplexes are very close (within experimental error; Fig. 2, left, and Table 1). As previously observed, thermal denaturation is quasi irreversible for both samples in the micromolar strand concentration range—no reassociation is observed upon cooling.11 Second, the kinetics of association are identical, within experimental error. These experiments were performed by starting from isolated strands and comparing the association of L- and D-TG4T and the L + D mixture by UV-isothermal experiments at 6 1C in 10 mM lithium cacodylate and 0.5 M KCl conditions as previously described.11,15 One observes a time-dependent increase in absorbance at 295 nm that reflects the association of the different strands. These profiles may be fitted as previously described11 and lead to similar kon values (Table 1). 12,16 L-DNA cannot form stable duplexes with D-DNA. We therefore decided to investigate if the two enantiomers are compatible within an intermolecular quadruplex or if chiral selection occurs.17 Three independent experiments suggest that L/D hybrid structures are at most marginal: (i) Kinetics analysis of G4 formation with D + L mixtures supports chiral selection: when equimolar concentrations Chem. Commun., 2011, 47, 5437–5439
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Fig. 2 Stability and kinetics of G4 formed by L- and D-TG4T. Left: UV melting profiles at 295 nm in 0.1 M NaCl (0.3 1C min1; note that the apparent Tm depends on the scan rate as dissociation is nearly irreversible at this strand concentration). Right: isothermal association at 6 1C in 0.5 M KCl. Bottom: kon values calculated for the L- and D-oligonucleotides as well as the L + D mixture using two different models (assuming the concentrations are additive (‘‘L + D’’ case) or not (‘‘(L) (D)’’).
Table 1 Comparison of L- and D-TG4T. Apparent melting temperatures are determined at 295 nm in 0.1 M or 0.5 M NaCl (no melting occurs in KCl) with a temperature gradient of 0.3 and 1 1C min1, respectively. kon values were obtained in 0.5 M KCl from isothermal association experiments. Apparent thermal temperatures are determined with 1–2 1C accuracy
Fig. 3 Enantiomeric exclusion in quadruplex assembly. Left: self exclusion of L- and D-strands into quadruplexes. ‘‘M’’ lanes correspond to oligo dTn migration markers (length in nucleotides provided on the left). The gel is revealed by UV shadow. Lane 1: conditions unfavourable for G4 formation, only single strands are seen. Lanes 2–5: preincubation in KCl favoring G4 formation (90 mM for L- and D-TG4T; 180 mM for D-T4G4T). Right: using a CCC (a cationic combtype copolymer),22 we demonstrate that a fluorescent L-DNA strand (0.5 mM strand concentration) may be ‘‘inserted’’ into a preformed tetramolecular L-quadruplex (arrow) but not D-quadruplex (both at 20 mM strand concentration). The gel is revealed by fluorescence (i.e., only L-TG4T fluo is seen, either single-stranded (ss) or incorporated into G4).
T1/2/1C 0.1 M Na+
T1/2/1C 0.5 M Na+
kon/M3 s1
58 57
67.5 67
(9.4 3.6) 1010 (8.4 1.6) 1010
Fig. 4 Principle of the strand exchange experiment.21,22 In the presence of CCC, one can incorporate the fluorescent strand into a preformed tetramolecular quadruplex. This may be evidenced by electrophoresis. One may compare the strand exchange efficiency with L- or D-fluorescent strands, to be incorporated in a L- or D-preformed quadruplex.
of D and L strands are present, the association proceeds with kinetics that suggest that concentrations are not additive. As the rate of this reaction is strongly dependent on strand concentration11,18 (with an experimentally determined experimental order of 3 to 4) this suggests that both strands associate independently from each other (Fig. 2, bottom). (ii) When mixing two strands of different lengths, such as TG4T and T4G4T, one expects to see 5 bands for a tetramolecular complex, corresponding to the different possibilities (four short strands, three short, one long, etc.)19 (Fig. 3, left). This is indeed the case when mixing two D-strands, while a very different situation (compare lanes 4 and 5) is observed for L-TG4T + D-T4G4T, in which two dominant bands are obtained with migrations corresponding to the ‘‘pure’’ species [L-TG4T]4 and [D-T4G4T]4. (iii) Finally, using a fluorescent L-TG4T strand, we demonstrate that a cationic comb-type copolymer (CCC) acting as a nucleic acid chaperone20,21 promotes its incorporation by strand exchange into a preformed [L-TG4T]4 quadruplex, but not into [D-TG4T]4 (Fig. 3, right and Fig. 4). A reciprocal experiment with a fluorescent D-TG4T-fluorescein strand gives
a ‘‘mirror’’ result (incorporation into a preformed D-quadruplex, but not L-quadruplex; not shown). Altogether, our results demonstrate that one can obtain left-handed L-DNA tetramolecular quadruplexes and that chiral selection may be achieved. Several applications may be proposed for L-DNA quadruplexes: (i) As a number of aptamers adopt quadruplex folds,23 it should be interesting to develop G4-based spiegelmers, providing extra resistance in a biological environment. (ii) L- vs. D-Quadruplexes may be tested for quadruplex ligand affinity. A number of G4 ligands such as telomestatin possess chiral centers, potentially allowing chiral discrimination,24 as found for duplex binders:25 comparing their affinities for both quadruplexes might give clues on the binding mode, as little discrimination should be expected for terminal stacking while groove binding and interactions with the loops may differ. (iii) Chiral selection is an advantage in nanotechnology as L- and D- quadruplexes may be assembled simultaneously with little or no ‘‘crosstalk’’. This is in contrast with unnatural nucleic acids such as LNA,26 PNA27 and 2 0 O methyl RNAs11 which have been reported to form G-quadruplexes but may form hybrid structures such as 2PNA–2DNA.
Oligonucleotide LD-
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Funding was provided by INSERM, Universite´ de Bordeaux, CNRS-PIR, Fondation pour la Recherche Me´dicale, Re´gion Aquitaine, Japan Science and Technology Agency (JST) and ANR-09-Blanc-0355. J.L.M. would also like to thank N. Sharp and W. Bell (Massive Dynamic, Boston, MA, USA) for alternative funding.
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