This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 5437–5439 5437 Cite this: Chem. Commun., 2011, 47, 5437–5439 A mirror-image tetramolecular DNA quadruplexw Phong Lan Thao Tran, ab Rui Moriyama, c Atsushi Maruyama, c Bernard Rayner ab 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 PCR 3 or microarray 4 probes, agents for enantiomeric separation 5 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 [TG 4 T] 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-2 0 - deoxy-L-guanosine and L-thymidine were dimethoxytritylated and further converted to the corresponding b-cyanoethylphosphor- amidites 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-TG 4 T 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-TG 4 T. 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 (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 temperatures 11 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-TG 4 T 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 described 11 and lead to similar k on values (Table 1). L-DNA cannot form stable duplexes with D-DNA. 12,16 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 Fig. 1 Evidence for G4 formation by L- and D-TG 4 T. 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 (À) to G4 formation (60 mM strand concentration). ‘‘M’’ lanes correspond to oligo dT n migration markers. Right: CD spectra of the L- and D- quadruplexes in 0.5 M KCl. a Universite ´ de Bordeaux, Laboratoire ARNA, F-33000 Bordeaux, France. E-mail: jean-louis.mergny@inserm.fr 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 ChemComm Dynamic Article Links www.rsc.org/chemcomm COMMUNICATION Downloaded by UNIV BORDEAUX 1-SCD-BIBLIO UNIVERSITAIRE on 27 April 2011 Published on 11 April 2011 on http://pubs.rsc.org | doi:10.1039/C1CC11293G View Online