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Cite this: Chem. Commun., 2014, 50, 605 Received 2nd October 2013, Accepted 4th November 2013
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Functional isoDNA aptamers: modified thrombin binding aptamers with a 2 0 -5 0 -linked sugarphosphate backbone (isoTBA)† Anita D. Gunjal, Moneesha Fernandes, Namrata Erande, P. R. Rajamohanan and Vaijayanti A. Kumar*
DOI: 10.1039/c3cc47569g www.rsc.org/chemcomm
The regioisomeric 30 -deoxy-20 -50 -linked thrombin binding DNA aptamers (isoTBAs) were chemically synthesized and their ability to form unimolecular anti-parallel G-quadruplexes in the presence of K+ ions was evaluated. These modified sequences retain the function of the native thrombin binding aptamer (TBA), exhibit better stability against exonuclease and are capable of slowing down the process of blood clotting.
The 30 -deoxy-20 -50 -linked isoDNA–isoRNA sequences (Fig. 1) are known to form duplexes with complementary RNA, but their thermal stability is lower for DNA:DNA/DNA:RNA or RNA:RNA duplexes.1 These non-genetic isoDNA oligomers are also known to be involved in triplex structures1,2 but higher order structures involving the isoDNA backbone remain unexplored. Higher order DNA structures such as those containing G-quadruplexes have been gaining increasing attention3 in the recent past because of their abundance in vivo, in telomeres, as targets for cancer treatments and also in nanotechnology.4 In this communication, we show, for the first time, that the homogeneous 2 0 -5 0 -linked isoDNA TBA sequence is capable of forming G-quadruplex structures. Further it is shown
that the 2 0 -5 0 -linked unnatural backbone retained the functional molecular recognition ability of the natural DNA aptamer. We selected a well-documented, G-quadruplex-forming 3 0 -5 0 linked TBA sequence4 5 0 -GGTTGGTGTGGTTGG-3 0 (TBA-1) as an example to study the structural topology of the 2 0 -5 0 -linked 50 -GGTTGGTGTGGTTGG-20 sequence (isoTBA-2). We envisaged isoDNA as a backbone candidate for the possible quadruplex formation considering the following points: (1) the 20 -50 linkages maintain an extended backbone geometry due to the anomeric effect and the O40 -C10 -C20 -O20 gauche effect on the substituted sugar, leading to the N-type sugar conformations,5 (2) in 30 deoxy-ribonucleosides, the guanine base orientation could be either syn or anti,6 (3) the 20 -50 -linkages are known to form stable loop structures in the hairpin DNA/RNA motifs,7 and (4) the isoDNA oligomers are relatively stable to exonuclease degradation.1 The first three points are requisite features of an intramolecular antiparallel folded quadruplex.3 The last point is important from the application perspective. TBA binds to a large protein, thrombin, involved in blood coagulation. The synthetic oligonucleotides, less susceptible to enzyme digestion, have potential applications in cardiovascular diseases.8 The synthetic oligonucleotide sequences and their characterization are shown in Table 1. Replacement of 30 -deoxythymidine in the TGT loop (T7 and T9 in isoTBA-2) by uridine gave UGU loopmodified sequence 5 0 -GGTTGGUGUGGTTGG-2 0 (isoTBA-3). The 3 0 -OH group might shift the equilibrium to a S-type sugar Table 1
TBA/isoTBA sequences of the study
Tm (1C)
Sequences
MALDI-TOF mass Calcd/obsd
Na+
K+
Thrombin (K+)a
Fig. 1 Genetic 3 0 -5 0 -linked DNA/RNA and non-genetic 2 0 -5 0 -linked isoDNA/isoRNA.
TBA-1 isoTBA-2 isoTBA-3
4726/4728 4726/4731 4730/4732
22.2 24.9 nd
52.0 37.1 45.0
22 (53) o10 (38) 13.2 (45)
Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India. E-mail: [email protected]; Tel: +91 2025902340 † Electronic supplementary information (ESI) available: Experimental conditions and spectral data figures. See DOI: 10.1039/c3cc47569g
TBA-1: 5 0 -GGTTGGTGTGGTTGG-3 0 , native 3 0 -5 0 phosphate-containing DNA; isoTBA-2: 5 0 -GGTTGGTGTGGTTGG-2 0 , 2 0 -5 0 phosphate-containing isoDNA; isoTBA-3: 5 0 -GGTTGGUGUGGTTGG-2 0 , 2 0 -5 0 phosphatecontaining isoDNA with T7 and T9 replaced by U. a Values in parentheses are obtained in the presence of thrombin and K+ ions.
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conformation in this case1 and impart rigidity and stability to the loop similar to that imparted by the N-type sugars in DNA.9 The most recent corrected crystal structure of thrombin complexed with TBA10 revealed an extended interface between thrombin exosite I and the two TT loops of the G-quadruplex and minimum interactions of thrombin with the TGT loop in the TBA. The sugar residues in the TT loop involved in interactions with thrombin and the guanine nucleosides involved in the G-quartet formation were therefore not modified in our design. The G-quadruplex formation for these sequences was studied by CD spectroscopy9,11 in the presence of added monovalent cations such as K+ (Fig. 2) and Na+ and their stability was determined as a function of temperature-dependent changes in CD amplitude at 295 nm (ESI,† Fig. S1 and Table 1). All the sequences exhibited intense maxima at 295 nm in the CD spectra, corresponding to the group-III antiparallel G-quadruplex topology9,11 in the presence of K+ ions (Fig. 2). The stability of the G-quadruplexes was followed by the change in the amplitude of the CD signal at 295 nm with temperature (ESI,† Fig. S1). The dependence of quadruplex stability on the monovalent cation used (Na+ or K+) was also studied and K+ highly favoured the quadruplex stability. The isoTBA-2 structure was less stable compared to the control TBA-1 in the presence of K+ (Tm = 37.1 1C and 52 1C respectively), but their stability was comparable in the presence of Na+ (Tm = 24.9 1C and 22.2 1C respectively). The replacement of TGT by UGU in the loop region (isoTBA-3) improved the stability of the quadruplex structure as seen by a positive change in Tm in the presence of K+ (Table 1). Thus, the loop geometry of the isomeric DNA also contributes to the quadruplex stability as found earlier for TBA-1.9 Hysteresis between the heating and cooling curves was found to be negligible in all the cases (ESI,† Fig. S2). These results were further supplemented by
Fig. 2 CD spectra of oligomers TBA-1, isoTBA-2 and isoTBA-3 at 5 mM strand concentration and buffer containing 100 mM KCl.
Fig. 3
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UV-Tm and hysteresis measurements (ESI,† Fig. S3).9 The Tms were also not affected at higher strand concentrations (20 mM) (ESI,† Fig. S4) indicating unimolecular folding into G-quadruplex form. The characteristic chemical shifts of imino protons involved in the H-bond formation between the guanines of the G-quartet of a quadruplex structure are observed between 11.5 and 12.5 ppm in the 1 H NMR spectrum.4,9 The imino proton chemical shifts for isoTBA-2 were found to be comparable to those for the control TBA-1, indicating the hydrogen-bonded quadruplex structure for both oligomers (ESI,† Fig. S5). TBA-1 displayed 1H NMR signals between 11.5 and 12.5 ppm at 4 1C even in the absence of K+, whereas isoTBA-2 did not show any inclination to fold into a quadruplex form in the absence of K+ (ESI,† Fig. S5, inset). The temperature-dependent changes in the spectra were then recorded in the presence of K+. As observed in the CD experiments at 295 nm, the 1H-NMR signals between 11.5 and 12.5 ppm slowly disappeared with increasing temperature. In the case of TBA-1 and isoTBA-2, the NMR signals were observed up to 50 1C and 33 1C, respectively (ESI,† Fig. S5). The temperatures, at which the disappearance of the H-bonded imino proton signals is observed, co-related with the CD-Tm values of the two oligomers (Table 1, Tm 52 1C and 37 1C, respectively) which could be expected with the loss of H-bonded imino protons of quadruplex structures near the Tm. The broad peaks between 10.5 and 11.1 ppm in the TBA-1 and isoTBA-2 spectra disappear at B20–25 1C in each case, much below the melting temperature obtained from CD experiments (52 1C and 37 1C respectively), suggesting that these may not be involved in the H-bonded quadruplex structure. It has been earlier demonstrated that thrombin can act as a molecular chaperone for the folding of TBA-1 into a G-quadruplex structure even in the absence of monovalent cations. Changes in CD signal amplitude at 295 nm upon addition of thrombin were used as a measure for this induced G-quadruplex formation at 4 1C.12 We performed similar CD experiments with TBA-1, isoTBA-2 and isoTBA-3 in the presence of increasing concentrations of thrombin at 4 1C (Fig. 3). We observed an increase in the CD signal amplitude for all the sequences upon incremental addition of thrombin (0.0 to 11.1 104 mmol).12 In the case of control TBA-1, a CD maximum at 295 nm was observed even in the absence of either thrombin or K+, revealing its propensity towards G-quadruplex structures as reported earlier12 and also as indicated by 1H NMR in the present studies (ESI,† Fig. S5, inset). In the case of isoTBA-2 and isoTBA-3, the characteristic quadruplex CD band at 295 nm was not observed in the absence of either thrombin or K+, (as seen also by the absence of imino proton signals in the 1H NMR
Changes in the CD signal at 295 nm upon addition of thrombin to TBA-1, isoTBA-2 and isoTBA-3 at 5 mM strand concentration in water in the absence of K+.
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spectrum in the absence of K+, ESI,† Fig. S5, inset). Isoelliptic points were observed at B280 nm and B255 nm in all the cases, indicating the two-state nature of the structural transition upon addition of thrombin alone.12 The stability of these induced G-quadruplexes in the absence of K+ was followed by the change in the amplitude of the CD signal at 295 nm with temperature. The strength of the TBA-1 G-quadruplex was the highest at Tm = 22 1C, followed by isoTBA-3 (Tm = 13 1C) and isoTBA-2 (Tm r 10 1C). The CD signal at 295 nm could be restored upon addition of K+ and the quadruplexes were found to be as stable as with K+ alone (Table 1). Changes in CD spectral amplitudes were not observed when serum albumin was used instead of thrombin in these experiments substantiating the specific role of thrombin in inducing the quadruplex structures12 (ESI,† Fig. S6). These results clearly point out the similarity in structural topology of isoTBA with TBA, which would allow the specific interactions with thrombin. To test the enzymatic stability of the isoTBA as compared to TBA, we subjected these aptamers to snake venom phosphodiesterase (SVPD) digestion (ESI,† Fig. S7).1 The 2 0 -5 0 -linked isoTBA-2 was found to be only 50% digested after 2 h compared to the control TBA-1 which was completely digested within 1 h. The observed stability of the isoDNA oligomer offers obvious advantages for applications in biological systems. We further investigated the inhibitory activity of the aptamers on thrombin-catalyzed conversion of fibrinogen to fibrin (clotting) by measuring the percent transmittance (% T) with time in the absence of potassium ions. The aptamer TBA-1 slowed down the coagulation with an induction time (time at which the transmittance starts decreasing due to coagulation) of 30 min, confirming its reported inhibitory activity (Fig. 4).13 The induction time for the isoDNA oligomers, isoTBA-2 and isoTBA-3 (15 min and 12 min respectively), was less than for TBA-1, but more than that in the absence of any aptamer. Thus, the isoTBA sequences hold similarity in structural topology capable of taking active part in the assigned biological function of the TBA, though with about 50% efficiency. In the presence of SVPD, as expected, the induction time for TBA-1 was reduced to 3 min compared to 6 min for the isoTBA-2 aptamer (ESI,† Fig. S8). Backbone modifications of TBA are reported to have profound effects on the structural topology of the quadruplexes formed. The folding patterns of an isosequential RNA-TBA sequence14 showed that in contrast to the unimolecular antiparallel G-quadruplex structure of TBA,4 the RNA-TBA oligomer formed a multimolecular parallel G-quadruplex.14 A mixed DNA–RNA backbone TBA
Fig. 4 Antithrombin activity measured by UV transmittance (T) in the presence of 0.037 mM TBA-1, isoTBA-2 and iso-TBA-3.
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sequence, depending on the position of the ribo- or deoxyribonucleotides in the sequence, either folded in DNA-like (unimolecular, antiparallel) or RNA-like (tetramolecular, parallel) quadruplex structures.15 The backbone element is thus the crucial governing entity for maintaining the structural topology and biological function of a given aptamer sequence. The results presented here show with conclusive spectroscopic evidence that in spite of the isomeric, homogeneous 20 -50 backbone, isoDNA does form stable quadruplex structures and also retains the molecular recognition ability of the large protein thrombin. In conclusion, we have demonstrated that the G-rich isoTBA sequence with an isoDNA backbone is capable of forming a G-quadruplex structure and exhibits similar antithrombin activity to that of the SELEX-derived TBA. This is the first example of a G-quadruplex with an isomeric 2 0 -5 0 -phosphate backbone in the literature. It is remarkable that in spite of the fact that the sequence was originally evolved to interact with thrombin for a 3 0 -5 0 -DNA backbone, its structural and functional properties were retained in a 2 0 -5 0 -isoDNA backbone. Financial support from the Council of Scientific and Industrial Research is gratefully acknowledged (GenCODE BSC0123).
Notes and references 1 T. L. Sheppard and R. C. Breslow, J. Am. Chem. Soc., 1996, 118, 9810–9811; T. P. Prakash, K.-E. Jung and C. Switzer, Chem. Commun., 1996, 1793–1795; K.-E. Jung and C. Switzer, J. Am. Chem. Soc., 1994, 116, 6059–6060; P. A. Giannaris and M. J. Dahma, Nucleic Acids Res., 1993, 20, 4742–4749; B. J. Premraj, S. Raja, N. S. Bhavesh, K. Shi, R. V. Hosur, M. Sundaralingam and N. Yathindra, Eur. J. Biochem., 2004, 271, 2956–2966; N. D. Erande, A. D. Gunjal, M. Fernandes and V. A. Kumar, Chem. Commun., 2011, 47, 4007–4009; N. Erande, A. D. Gunjal, M. Fernandes, R. Gonnade and V. A. Kumar, Org. Biomol. Chem., 2013, 11, 746–757. 2 H. Sawai, J. Seki and H. Ozaki, J. Biomol. Struct. Dyn., 1996, 13, 1043–1051; M. J. Dahma and A. Noronha, Nucleic Acids Res., 1998, 26, 5152–5156. 3 S. Neidle, S. Balasubramanian, S. F. Campbell, M. Clore and D. M. Lilley, Quadruplex Nucleic Acids, RSC publications, Cambridge, UK, 2006; A. Avino, C. Fabrega, M. Tintore and R. Eritja, Curr. Pharm. Des., 2012, 18, 2036–2047; K. Y. Wang, S. McCurdy, R. G. Shea, S. Swaminathan and P. H. Bolton, Biochemistry, 1993, 32, 1899–1904; W. O. Tucker, K. T. Shum and J. A. Tanner, Curr. Pharm. Des., 2012, 18, 2014–2026. 4 A. Rajendran, M. Endo, K. Hidaka, P. L. Thao Tran, J.-L. Mergny and H. Sugiyama, Nucleic Acids Res., 2013, 41, 8738–8747; Guanine Quartets: Structure and Application, ed. L. Spindler and W. Fritzsche, RSC publications, Cambridge, UK, 2013. 5 M. Polak, M. Manoharan, G. B. Inamati and J. Plavec, Nucleic Acids Res., 2003, 31, 2066–2076. 6 A. Kumar, S. B. Katti, H. Rosemeyer and F. Seela, Nucleosides Nucleotides, 1996, 15, 1595–1601. 7 R. N. Hannoush and M. J. Damha, J. Am. Chem. Soc., 2001, 123, 12368–12374. 8 W. Kuliczkowski, J. Floyd, A. Malinin and V. Serebruany, Expert Rev. Cardiovasc. Ther., 2010, 8, 503–507; S. Nagatoishi, Y. Tanaka and K. Tsumoto, Biochem. Biophys. Res. Commun., 2007, 352, 812–817. 9 A. Avino, S. Mazzini, R. Ferreira, R. Gargallo, V. E. Marquez and R. Eritja, Bioorg. Med. Chem., 2012, 20, 4186–4193. 10 I. R. Krauss, A. Merlino, A. Randazzo, E. Novellino, M. Lelio and F. Sica, Nucleic Acids Res., 2012, 40, 8119–8128. 11 A. I. Karsisiotis, N. M. Hessari, E. Novellino, G. P. Spada, A. Randazzo and M. W. da Silva, Angew. Chem., Int. Ed., 2011, 50, 10645–10648. 12 E. Baldrich and C. K. O’Sullivan, Anal. Biochem., 2005, 341, 194–197; S. Nagatoishi, Y. Tanaka and K. Tsumoto, Biochem. Biophys. Res. Commun., 2007, 352, 812–817. 13 S. Uehara, N. Shimada, Y. Takeda, Y. Koyama, Y. Takei, H. Ando, S. Satoh, A. Uno and K. Sakurai, Bull. Chem. Soc. Jpn., 2008, 81, 1485–1491. 14 A. Joachimi, A. Benz and J. S. Hartig, Bioorg. Med. Chem., 2009, 17, 6811–6815. 15 C.-F. Tang and R. H. Shafer, J. Am. Chem. Soc., 2006, 128, 5966–5973.
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Cite this: Chem. Commun., 2014, 50, 605 Received 2nd October 2013, Accepted 4th November 2013
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Functional isoDNA aptamers: modified thrombin binding aptamers with a 2 0 -5 0 -linked sugarphosphate backbone (isoTBA)† Anita D. Gunjal, Moneesha Fernandes, Namrata Erande, P. R. Rajamohanan and Vaijayanti A. Kumar*
DOI: 10.1039/c3cc47569g www.rsc.org/chemcomm
The regioisomeric 30 -deoxy-20 -50 -linked thrombin binding DNA aptamers (isoTBAs) were chemically synthesized and their ability to form unimolecular anti-parallel G-quadruplexes in the presence of K+ ions was evaluated. These modified sequences retain the function of the native thrombin binding aptamer (TBA), exhibit better stability against exonuclease and are capable of slowing down the process of blood clotting.
The 30 -deoxy-20 -50 -linked isoDNA–isoRNA sequences (Fig. 1) are known to form duplexes with complementary RNA, but their thermal stability is lower for DNA:DNA/DNA:RNA or RNA:RNA duplexes.1 These non-genetic isoDNA oligomers are also known to be involved in triplex structures1,2 but higher order structures involving the isoDNA backbone remain unexplored. Higher order DNA structures such as those containing G-quadruplexes have been gaining increasing attention3 in the recent past because of their abundance in vivo, in telomeres, as targets for cancer treatments and also in nanotechnology.4 In this communication, we show, for the first time, that the homogeneous 2 0 -5 0 -linked isoDNA TBA sequence is capable of forming G-quadruplex structures. Further it is shown
that the 2 0 -5 0 -linked unnatural backbone retained the functional molecular recognition ability of the natural DNA aptamer. We selected a well-documented, G-quadruplex-forming 3 0 -5 0 linked TBA sequence4 5 0 -GGTTGGTGTGGTTGG-3 0 (TBA-1) as an example to study the structural topology of the 2 0 -5 0 -linked 50 -GGTTGGTGTGGTTGG-20 sequence (isoTBA-2). We envisaged isoDNA as a backbone candidate for the possible quadruplex formation considering the following points: (1) the 20 -50 linkages maintain an extended backbone geometry due to the anomeric effect and the O40 -C10 -C20 -O20 gauche effect on the substituted sugar, leading to the N-type sugar conformations,5 (2) in 30 deoxy-ribonucleosides, the guanine base orientation could be either syn or anti,6 (3) the 20 -50 -linkages are known to form stable loop structures in the hairpin DNA/RNA motifs,7 and (4) the isoDNA oligomers are relatively stable to exonuclease degradation.1 The first three points are requisite features of an intramolecular antiparallel folded quadruplex.3 The last point is important from the application perspective. TBA binds to a large protein, thrombin, involved in blood coagulation. The synthetic oligonucleotides, less susceptible to enzyme digestion, have potential applications in cardiovascular diseases.8 The synthetic oligonucleotide sequences and their characterization are shown in Table 1. Replacement of 30 -deoxythymidine in the TGT loop (T7 and T9 in isoTBA-2) by uridine gave UGU loopmodified sequence 5 0 -GGTTGGUGUGGTTGG-2 0 (isoTBA-3). The 3 0 -OH group might shift the equilibrium to a S-type sugar Table 1
TBA/isoTBA sequences of the study
Tm (1C)
Sequences
MALDI-TOF mass Calcd/obsd
Na+
K+
Thrombin (K+)a
Fig. 1 Genetic 3 0 -5 0 -linked DNA/RNA and non-genetic 2 0 -5 0 -linked isoDNA/isoRNA.
TBA-1 isoTBA-2 isoTBA-3
4726/4728 4726/4731 4730/4732
22.2 24.9 nd
52.0 37.1 45.0
22 (53) o10 (38) 13.2 (45)
Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India. E-mail: [email protected]; Tel: +91 2025902340 † Electronic supplementary information (ESI) available: Experimental conditions and spectral data figures. See DOI: 10.1039/c3cc47569g
TBA-1: 5 0 -GGTTGGTGTGGTTGG-3 0 , native 3 0 -5 0 phosphate-containing DNA; isoTBA-2: 5 0 -GGTTGGTGTGGTTGG-2 0 , 2 0 -5 0 phosphate-containing isoDNA; isoTBA-3: 5 0 -GGTTGGUGUGGTTGG-2 0 , 2 0 -5 0 phosphatecontaining isoDNA with T7 and T9 replaced by U. a Values in parentheses are obtained in the presence of thrombin and K+ ions.
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conformation in this case1 and impart rigidity and stability to the loop similar to that imparted by the N-type sugars in DNA.9 The most recent corrected crystal structure of thrombin complexed with TBA10 revealed an extended interface between thrombin exosite I and the two TT loops of the G-quadruplex and minimum interactions of thrombin with the TGT loop in the TBA. The sugar residues in the TT loop involved in interactions with thrombin and the guanine nucleosides involved in the G-quartet formation were therefore not modified in our design. The G-quadruplex formation for these sequences was studied by CD spectroscopy9,11 in the presence of added monovalent cations such as K+ (Fig. 2) and Na+ and their stability was determined as a function of temperature-dependent changes in CD amplitude at 295 nm (ESI,† Fig. S1 and Table 1). All the sequences exhibited intense maxima at 295 nm in the CD spectra, corresponding to the group-III antiparallel G-quadruplex topology9,11 in the presence of K+ ions (Fig. 2). The stability of the G-quadruplexes was followed by the change in the amplitude of the CD signal at 295 nm with temperature (ESI,† Fig. S1). The dependence of quadruplex stability on the monovalent cation used (Na+ or K+) was also studied and K+ highly favoured the quadruplex stability. The isoTBA-2 structure was less stable compared to the control TBA-1 in the presence of K+ (Tm = 37.1 1C and 52 1C respectively), but their stability was comparable in the presence of Na+ (Tm = 24.9 1C and 22.2 1C respectively). The replacement of TGT by UGU in the loop region (isoTBA-3) improved the stability of the quadruplex structure as seen by a positive change in Tm in the presence of K+ (Table 1). Thus, the loop geometry of the isomeric DNA also contributes to the quadruplex stability as found earlier for TBA-1.9 Hysteresis between the heating and cooling curves was found to be negligible in all the cases (ESI,† Fig. S2). These results were further supplemented by
Fig. 2 CD spectra of oligomers TBA-1, isoTBA-2 and isoTBA-3 at 5 mM strand concentration and buffer containing 100 mM KCl.
Fig. 3
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UV-Tm and hysteresis measurements (ESI,† Fig. S3).9 The Tms were also not affected at higher strand concentrations (20 mM) (ESI,† Fig. S4) indicating unimolecular folding into G-quadruplex form. The characteristic chemical shifts of imino protons involved in the H-bond formation between the guanines of the G-quartet of a quadruplex structure are observed between 11.5 and 12.5 ppm in the 1 H NMR spectrum.4,9 The imino proton chemical shifts for isoTBA-2 were found to be comparable to those for the control TBA-1, indicating the hydrogen-bonded quadruplex structure for both oligomers (ESI,† Fig. S5). TBA-1 displayed 1H NMR signals between 11.5 and 12.5 ppm at 4 1C even in the absence of K+, whereas isoTBA-2 did not show any inclination to fold into a quadruplex form in the absence of K+ (ESI,† Fig. S5, inset). The temperature-dependent changes in the spectra were then recorded in the presence of K+. As observed in the CD experiments at 295 nm, the 1H-NMR signals between 11.5 and 12.5 ppm slowly disappeared with increasing temperature. In the case of TBA-1 and isoTBA-2, the NMR signals were observed up to 50 1C and 33 1C, respectively (ESI,† Fig. S5). The temperatures, at which the disappearance of the H-bonded imino proton signals is observed, co-related with the CD-Tm values of the two oligomers (Table 1, Tm 52 1C and 37 1C, respectively) which could be expected with the loss of H-bonded imino protons of quadruplex structures near the Tm. The broad peaks between 10.5 and 11.1 ppm in the TBA-1 and isoTBA-2 spectra disappear at B20–25 1C in each case, much below the melting temperature obtained from CD experiments (52 1C and 37 1C respectively), suggesting that these may not be involved in the H-bonded quadruplex structure. It has been earlier demonstrated that thrombin can act as a molecular chaperone for the folding of TBA-1 into a G-quadruplex structure even in the absence of monovalent cations. Changes in CD signal amplitude at 295 nm upon addition of thrombin were used as a measure for this induced G-quadruplex formation at 4 1C.12 We performed similar CD experiments with TBA-1, isoTBA-2 and isoTBA-3 in the presence of increasing concentrations of thrombin at 4 1C (Fig. 3). We observed an increase in the CD signal amplitude for all the sequences upon incremental addition of thrombin (0.0 to 11.1 104 mmol).12 In the case of control TBA-1, a CD maximum at 295 nm was observed even in the absence of either thrombin or K+, revealing its propensity towards G-quadruplex structures as reported earlier12 and also as indicated by 1H NMR in the present studies (ESI,† Fig. S5, inset). In the case of isoTBA-2 and isoTBA-3, the characteristic quadruplex CD band at 295 nm was not observed in the absence of either thrombin or K+, (as seen also by the absence of imino proton signals in the 1H NMR
Changes in the CD signal at 295 nm upon addition of thrombin to TBA-1, isoTBA-2 and isoTBA-3 at 5 mM strand concentration in water in the absence of K+.
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spectrum in the absence of K+, ESI,† Fig. S5, inset). Isoelliptic points were observed at B280 nm and B255 nm in all the cases, indicating the two-state nature of the structural transition upon addition of thrombin alone.12 The stability of these induced G-quadruplexes in the absence of K+ was followed by the change in the amplitude of the CD signal at 295 nm with temperature. The strength of the TBA-1 G-quadruplex was the highest at Tm = 22 1C, followed by isoTBA-3 (Tm = 13 1C) and isoTBA-2 (Tm r 10 1C). The CD signal at 295 nm could be restored upon addition of K+ and the quadruplexes were found to be as stable as with K+ alone (Table 1). Changes in CD spectral amplitudes were not observed when serum albumin was used instead of thrombin in these experiments substantiating the specific role of thrombin in inducing the quadruplex structures12 (ESI,† Fig. S6). These results clearly point out the similarity in structural topology of isoTBA with TBA, which would allow the specific interactions with thrombin. To test the enzymatic stability of the isoTBA as compared to TBA, we subjected these aptamers to snake venom phosphodiesterase (SVPD) digestion (ESI,† Fig. S7).1 The 2 0 -5 0 -linked isoTBA-2 was found to be only 50% digested after 2 h compared to the control TBA-1 which was completely digested within 1 h. The observed stability of the isoDNA oligomer offers obvious advantages for applications in biological systems. We further investigated the inhibitory activity of the aptamers on thrombin-catalyzed conversion of fibrinogen to fibrin (clotting) by measuring the percent transmittance (% T) with time in the absence of potassium ions. The aptamer TBA-1 slowed down the coagulation with an induction time (time at which the transmittance starts decreasing due to coagulation) of 30 min, confirming its reported inhibitory activity (Fig. 4).13 The induction time for the isoDNA oligomers, isoTBA-2 and isoTBA-3 (15 min and 12 min respectively), was less than for TBA-1, but more than that in the absence of any aptamer. Thus, the isoTBA sequences hold similarity in structural topology capable of taking active part in the assigned biological function of the TBA, though with about 50% efficiency. In the presence of SVPD, as expected, the induction time for TBA-1 was reduced to 3 min compared to 6 min for the isoTBA-2 aptamer (ESI,† Fig. S8). Backbone modifications of TBA are reported to have profound effects on the structural topology of the quadruplexes formed. The folding patterns of an isosequential RNA-TBA sequence14 showed that in contrast to the unimolecular antiparallel G-quadruplex structure of TBA,4 the RNA-TBA oligomer formed a multimolecular parallel G-quadruplex.14 A mixed DNA–RNA backbone TBA
Fig. 4 Antithrombin activity measured by UV transmittance (T) in the presence of 0.037 mM TBA-1, isoTBA-2 and iso-TBA-3.
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sequence, depending on the position of the ribo- or deoxyribonucleotides in the sequence, either folded in DNA-like (unimolecular, antiparallel) or RNA-like (tetramolecular, parallel) quadruplex structures.15 The backbone element is thus the crucial governing entity for maintaining the structural topology and biological function of a given aptamer sequence. The results presented here show with conclusive spectroscopic evidence that in spite of the isomeric, homogeneous 20 -50 backbone, isoDNA does form stable quadruplex structures and also retains the molecular recognition ability of the large protein thrombin. In conclusion, we have demonstrated that the G-rich isoTBA sequence with an isoDNA backbone is capable of forming a G-quadruplex structure and exhibits similar antithrombin activity to that of the SELEX-derived TBA. This is the first example of a G-quadruplex with an isomeric 2 0 -5 0 -phosphate backbone in the literature. It is remarkable that in spite of the fact that the sequence was originally evolved to interact with thrombin for a 3 0 -5 0 -DNA backbone, its structural and functional properties were retained in a 2 0 -5 0 -isoDNA backbone. Financial support from the Council of Scientific and Industrial Research is gratefully acknowledged (GenCODE BSC0123).
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