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8–17 DNAzyme modified with purine analogs in its catalytic core: The conservation of the five-membered moieties of purine residues
Bioorganic & Medicinal Chemistry Letters 22 (2012) 4238–4241
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
8–17 DNAzyme modified with purine analogs in its catalytic core: The conservation of the five-membered moieties of purine residues Wei Rong a, Liang Xu b, Yang Liu b, Jianping Yu a,⇑, Ying Zhou a, Keliang Liu b, Junlin He b,⇑ a b
College of Life Science, Guizhou University, Guiyang 550025, China Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
a r t i c l e
i n f o
Article history: Received 10 April 2012 Revised 8 May 2012 Accepted 9 May 2012 Available online 17 May 2012 Keywords: 8–17 DNAzyme Purine analogs Catalytic cleavage Chemical modification 7-Nitrogen atom
a b s t r a c t 8–17 DNAzyme is characterized by its recurrence in different in vitro selections and versatile cleavage sites, leading to extensive studies on its structural properties and applications. We evaluated the purine residues (A6, G7, G11, A12, G14, and A15) in the catalytic core of 8–17 DNAzyme of their five-membered ring moiety with purine analogs 1–5 to have an insight into the conservation of the residues at the level of functional groups. The 7-nitrogen atom in the AGC loop was demonstrated to be strictly conserved for the cleavage reaction. But such modifications exerted favorable effect at G11 of the base-pair stem and A12 in the single-strand loop, directing toward more efficient DNAzymes. Even the most conserved G14 could tolerate such modifications. These results demonstrated that chemical modification on the functional groups is a feasible approach to gain an insight into the structural requirement in the catalytic reaction of DNAzymes. It also provided modification sites for introduction of signaling molecules used for mechanistic and folding studies of 8–17 DNAzyme. Ó 2012 Elsevier Ltd. All rights reserved.
Since the appearance of small artificial DNAzymes 10–23 and 8–17 and the development of their wide-broad applications, many new DNAzyme entities have also been selected under specific conditions.1–4 Interestingly, numerous 8–17 DNAzyme variants (e.g., 17E and Mg5) have been selected independently from different in vitro selection experiments.5–9 Their catalytic cleavage activity and specific ion-dependence has led to a new class of metal biosensors.10–12 Such recurrence of 8–17 motifs and versatile cleavage sites13 initiated many studies on its catalytic mechanism and folding,14 either with residue replacement,15 spectroscopic methods,16,17 or contact photo-crosslinking experiments.18,19 In the catalytic core, all of them share a highly conserved AGC triloop, a Watson–Crick base-pair stem and the single-strand loop of four or five residues (Fig. 1).13 Each residue was supposed to offer base stacking and several functional groups to form a complex hydrogen-bond net responsible for the active conformation. Although we could not yet conclude with a precise catalytic site or a role of specific residues in the catalytic core of 8–17, extensive studies are still being conducted to gain an insight about its structure and mechanisms for a better design of metal biosensors and other applications. Considering that the residues at each position in the catalytic core is the result of repeated selection and the positive role of base stacking, we started the chemical modification on specific functional groups around nucleobases in the catalytic core, in ⇑ Corresponding authors. Tel.: +86 851 385 4677; fax: +86 851 385 6374 (J.Y.); tel./fax: +86 10 6821 1656 (J.H.). E-mail addresses: [email protected] (J. Yu), [email protected] (J. He). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.05.044
3'-TCC TAG AT rG rA CCG AGG TA-5' 2.1
5'-AGG ATC TA T 3
A
GGC TCC AT-3'
G 14 C C G AC G G 6A C G C 7
8-17 DNAzyme
8
Figure 1. The 8–17 DNAzyme designed against a DNA–RNA–DNA chimeric substrate. The arrow showed the cleavage site between diribonucleotide rA-rG. Bold letters indicated the most conserved purine residues.
order to have a better understanding of the conservation of nucleobases at the level of the functional groups and help the construction of more sensitive deoxyribozyme-based metal biosensors. Of the three structural domains in the catalytic core of 8–17 DNAzyme, there are eight purine residues, including three most conserved purine residues A6, G7, and G14, three guanine residues (G5, G10, and G11) in the base-pair stem, and A12 and A15 in the single-strand turn near the cleavage site. In this work, we present the evaluation of the five-membered ring moiety of these adenine and guanine residues except G5 and G10. The unnatural nucleoside analogs 1–520–22 (Fig. 2) with no changes on the Watson–Crick base-pairing functional groups were selected. By comparing with parent 8–17 DNAzyme, the contribution of 7-N atom as well as
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W. Rong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4238–4241
O
O
HN
HN N O
H2N HO
N
H2N HO
OH 1
HO
N
N O
HO
OH 4
N
OH 3 NH2
NH2
N
N N
N N O
HO
NH2
HN N O
N
N
OH 2
O
H2N
NH2
N N O
N
OH 5
Figure 2. Purine analogs used for the modification on purine residues in the catalytic core of 8–17 DNAzyme.
the five-membered ring moiety of purine residues was evaluated. Their phosphoramidites were prepared as previously reported (the preparation of phosphoramidite of 4 was presented in Supplementary data). 8–17 DNAzyme was designed against the sequence from the overlapping vpr and tat reading frames of HIV-1,23 and the target sequence was synthesized as a chimeric DNA–RNA–DNA, in which GA diribonucleotide was the cleavage site. 8–17 and its modified analogs were synthesized with conventional solid-phase synthesis and purified with denaturing PAGE (8% urea). All of them were characterized by MALDI-TOF (Table 1). Thermal measure-
Table 1 Characterization results of oligodeoxynucleotides by MALDI-TOF a
DNAzymes
Sequences
8-17DZ00
agg atc taT CCG AGC CGG ACG Agg ctc cat agg atc taT CCG A1C CGG ACG Agg ctc cat agg atc taT CCG A2C CGG ACG Agg ctc cat agg atc taT CCG 3GC CGG ACG Agg ctc cat agg atc taT CCG A4C CGG ACG Agg ctc cat agg atc taT CCG 5GC CGG ACG Agg ctc cat agg atc taT CCG AGC CG1 ACG Agg ctc cat agg atc taT CCG AGC CG2 ACG Agg ctc cat agg atc taT CCG AGC CG4 ACG Agg ctc cat agg atc taT CCG AGC CGG 3CG Agg ctc cat agg atc taT CCG AGC CGG 5CG Agg ctc cat agg atc taT CCG AGC CGG AC1 Agg ctc cat agg atc taT CCG AGC CGG AC2 Agg ctc cat agg atc taT CCG AGC CGG AC4 Agg ctc cat agg atc taT CCG AGC CGG ACG 3gg ctc cat agg atc taT CCG AGC CGG ACG 5gg ctc cat atg gag cca gta gat cct
8-17DZ01 8-17DZ02 8-17DZ03 8-17DZ04 8-17DZ05 8-17DZ06 8-17DZ07 8-17DZ08 8-17DZ09 8-17DZ10 8-17DZ11 8-17DZ12 8-17DZ13 8-17DZ14 8-17DZ15 D18
MS (calcd)
MS (found)
9212.4
9212.1
9211.4
9217.8
9212.4
9212.1
9212.4
9212.7
9269.2
9266.7
9269.6
9268.2
9211.4
9215.8
9212.4
9211.1
9269.6
9268.7
9212.4
9213.9
9269.6
9268.4
9211.4
9215.8
9212.4
9213.1
9269.6
9269.9
9212.4
9216.5
9269.6
9269.8
5521.0
5520.9
a The lowercase letters constitute both recognition arms, and the uppercase letters represent the catalytic core of 8–17 DNAzyme.
ment on the complexes between DNAzyme and its corresponding full-DNA substrate 50 -d(ATG GAG CCA GTA GAT CCT)-30 (D18, Table 1) in the reaction buffer indicated that all the modifications did not results in significant changes on thermal stabilities of the complexes (Supplementary data). The DNAzyme–substrate complex was formed in the reaction buffer. The cleavage activity of 8–17 and its analogs was determined under single-turnover conditions in the presence of 20 mM Mg2+, with DNAzymes of 100-fold more than the 32P-labeled substrate (Fig. 1). In addition to its repeated appearance in 8–17 variants, AGC loop has also been demonstrated to be highly conserved through mutagenesis with canonical and non-canonical residues, their functional groups, such as amino, keto, and 7-N atom are essential for the catalytic activity. In our evaluation system for modified 8– 17, as shown in Table 2, the same phenomenon was also observed.15 When the 7-N atom of G7 was deleted through the introduction of compound 1 at G7 in 8-17DZ01, a decreased kobs was obtained. Even a position shift of this nitrogen atom by compound 2 in 8-17DZ02 was not allowed, complete abolishment of cleavage activity was observed. Similar effect was produced when the 7-N atom of A6 was shifted to 8-position with compound 3 in 817DZ03. These observations indicated that A6 and G7 were probably located very close to the catalytic center, all functional groups around the two nucleobases might completely fold into the catalytic conformation, either by forming hydrogen-bonding network or positioning metal ions. In other words, the spacial distribution of these functional groups has been best defined by A6 and G7 as the repeated in vitro selection result. Further introduction of amino group at this loop with compound 4 or 5 led to new interactions accompanied by a complete loss of activity. This result further confirmed the absolute importance of the AGC loop in forming the catalytic conformation at the level of functional groups. The three base-pair stem has been supposed to play a structural role, offering stabilization for the catalytic conformation, based on the fact that G–C and A–T base pairs are well-tolerated, even mismatched base pairs were found in 8–17 variants. Especially, this stem could be replaced by T–Hg–T pair, with which mercuric ion switches on the stem formation and subsequently the cleavage reaction.24 Combining with fluorescent molecule labeling at the recognition arms, such 8–17 variants have been used as the biosensors for Hg2+. Based on these researches, the base pair in the stem was hypothesized to be located inside the helix, and their functional groups might not be involved in the catalytic conformation directly. Indeed, removal of 7-N atom by compound 1 at G11 did not produce significant effect on reaction rate (8-17DZ06, kobs = 0.0077 ± 0.0007 min1). However, when this 7-N was shifted to 8-position by compound 2 at G11, about twofold increase in reaction rate of 8-17DZ07 (kobs = 0.0131 ± 0.0005 min1) was produced. This slight improvement indicated that the five-membered
Table 2 kobs of 8–17 DNAzyme and its modified analogs measured under single-turnover conditionsa No.
kobs (min1)
No.
kobs (min1)
8-17DZ00 8-17DZ01 8-17DZ02 8-17DZ03 8-17DZ04 8-17DZ05 8-17DZ06 8-17DZ07
0.0064 ± 0.00055 0.0025 ± 0.00006 NDb ND ND ND 0.0077 ± 0.0007 0.0131 ± 0.0005
8-17DZ08 8-17DZ09 8-17DZ10 8-17DZ11 8-17DZ12 8-17DZ13 8-17DZ14 8-17DZ15
0.0103 ± 0.0010 0.0039 ± 0.00036 0.0101 ± 0.00105 0.0019 ± 0.00017 0.0098 ± 0.0013 0.0062 ± 0.0005 0.0014 ± 0.0001 <0.0001
a The cleavage reaction of DNAzyme (2 lM) on the 32P-labeled substrate (20 nM) were conducted in the buffer (50 mM Tris–HCl, 20 mM Mg2+) at 37 °C (more details see Supplementary data). b Not detected under present conditions.
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W. Rong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4238–4241
When G14 of 8–17 was further evaluated with compound 1 or 2, the kobs of new DNAzymes 8-17DZ11 (kobs = 0.0019 ± 0.00017 min1) and 8-17DZ12 (kobs = 0.0098 ± 0.0013 min1) demonstrated that 7-N and 8-N atoms at G14 were helpful for the cleavage reaction. Further perturbation with an extra amino group by compound 4 in this position could not offer more favorable effect on the reaction (8-17DZ13, kobs = 0.0062 ± 0.0005 min1). The cleavage pattern of these three DNAzymes within 300 min was shown in Figure 3B. This result told us that G14 is highly conserved as an integrate nucleobase, and its functional group distribution could be improved by functional group modification. For the adenine residues A12 and A15, mutations with compound 3 resulted in loss of activity, as shown in Table 2 (kobs = 0.0039 ± 0.00036 min1 for 8-17DZ09, and kobs = 0.0014 ± 0.0001 min1 for 8-17DZ14, respectively), it indicated that their 7-N atom was involved in the catalytic reaction. More adverse effect was observed for A15 when its 7-N was deleted. But the extra amino group by compound 5 in these two positions resulted in opposite effect. At A12, this extra amino group was beneficial for the reaction (8-17DZ10, kobs = 0.0101 ± 0.00105 min1), while it was deleterious for A15 (8-17DZ15). The cleavage behavior of these DNAzymes within 300 min was compared in Figure 3C. Based on the residue replacement and modification of functional groups on the catalytic core of 8–17 DNAzyme, we learnt that the residue conservation is determined by both base stacking and the interactions derived from functional groups. Chemical modifications on functional groups of specific nucleobases could give a more detailed picture of the residue involvement in the cleavage reaction. The two purine residues in the AGC loop was absolutely conserved at the level of functional groups, while the five-membered moiety of the conserved G14 could be modified with positive effect on the cleavage behavior. Based on the effect of compound 4 at G11 and A12, these two positions were the possible sites for introduction of signaling molecules used in the research of mechanism and folding of 8–17, through the 7-positioned amino group of compound 4. By this way, favorable modifications could be selected on the level of functional groups, either for more detailed understanding of the structural requirement in the catalytic reaction of DNAzymes, or for more efficient versions of DNAzymes. Acknowledgments This research was supported by National Natural Science Foundation of China 21072229 and Beijing Natural Science Foundation 7123223. Figure 3. The effect of purine analogs incorporated into 8–17 DNAzyme on the cleavage reactions: A, G11 replaced by 1 (8-17DZ06, d), 2 (8-17DZ07, N), 4 (817DZ08, ), parent 8–17 (j); B, G14 replaced with 1 (8-17DZ11, s), 2 (8-17DZ12, 4), 4 (8-17DZ13, }); C, A12 replaced by 3 (8-17DZ09,q) and 5 (8-17DZ10,w), A15 replaced by 3 (8-17DZ14,5), and 5 (8-17DZ15,.), respectively.
moiety of G11 might be involved in the cleavage reaction by its 7-N atom, and 8-N atom is a little more favorable for this positive effect than 7-N atom. Further introduction of compound 4 remained almost the same rate increase. Their cleavage pattern within 300 min was shown in Figure 3A. The effect of compound 4 at G11 and the extra amino group offers a potential spot for labeling 8–17 DNAzyme with signaling molecules in the studies of folding of 8–17 DNAzyme.17 The third domain in 8–17 DNAzyme is the single-strand turn close to the cleavage site, it differs from each other between its variants, ACGA in 8–17, TCGAA in 17E, and ACGAA in Mg5. C13G14 are the most conserved residues, any substitution with other three canonical residues led to deleterious effect on the cleavage activity.
Supplementary data Supplementary data (the preparation of phosphoramidite of compound 4 and Tm of the complexes between DNAzymes with the corresponding full-DNA substrate) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bmcl.2012.05.044. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262. Sekhon, G. S.; Sen, D. Biochemistry 2009, 48, 6335. Kandadai, S. A.; Mok, W. W. K.; Ali, Md. M.; Li, Y. Biochemistry 2009, 48, 7383. Schlosser, K.; Gu, J.; Lam, J. C. F.; Li, Y. Nucleic Acids Res. 2008, 36, 4768. Schlosser, K.; Li, Y. Nucleic Acids Res. 2009, 37, 413. Li, J.; Zheng, W.; Kwon, A. H.; Lu, Y. Nucleic Acids Res. 2000, 28, 481. Faulhammer, D.; Famulok, M. Angew. Chem., Int. Ed. 1996, 35, 2837. Faulhammer, D.; Famulok, M. J. Mol. Biol. 1997, 269, 188. Cruz, R. P. G.; Withers, J. B.; Li, Y. Chem. Biol. 2004, 11, 57. Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642. Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 7587. Teller, C.; Shimron, S.; Willner, I. Anal. Chem. 2009, 81, 9114.
W. Rong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4238–4241 13. 14. 15. 16. 17. 18. 19. 20. 21.
Brown, A. K.; Li, J.; Pavot, C. M.-B.; Lu, Y. Biochemistry 2003, 42, 7152. Leung, E. K. Y.; Sen, D. Chem. Biol. 2007, 14, 41. Peracchi, A.; Bonaccio, M.; Clerici, M. J. Mol. Biol. 2005, 352, 783. Lee, N. K.; Koh, H. R.; Han, K. Y.; Kim, S. K. J. Am. Chem. Soc. 2007, 129, 15526. Mazumdar, D.; Nagraj, N.; Kim, H.-K.; Meng, X.; Brown, A. K.; Sun, Q.; Li, W.; Lu, Y. J. Am. Chem. Soc. 2009, 131, 5506. Liu, Y.; Sen, D. J. Mol. Biol. 2008, 381, 845. Sekhon, G. S.; Sen, D. Biochemistry 2010, 49, 9072. Seela, F.; Westermann, B.; Bindig, U. J. Chem. Soc., Perkin Trans. I 1988, 697. Froehler, B. C. In Methods in Molecular Biology; Agrawal, E. S., Ed.; Humana Press, 1994; Vol. 20, p 63.
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22. He, J.; Zhang, D.; Wang, Q.; Wei, X.; Cheng, M.; Liu, K. Org. Biomol. Chem. 2011, 9, 5728. 23. Mitsuyasu, R. T.; Merigan, T. C.; Carr, A.; Zack, J. A.; Winters, M. A.; Workman, C.; Bloch, M.; Lalezari, J.; Becker, S.; Thornton, L.; Akil, B.; Khanlou, H.; Finlayson, R.; McFarlane, R.; Smith, Don E.; Garsia, R.; Ma, D.; Law, M.; Murray, J. M.; Von Kalle, C.; Ely, J. A.; Patino, S. M.; Knop, A. E.; Wong, P.; Todd, A. V.; Haughton, M.; Fuery, C.; Macpherson, J. L.; Symonds, G. P.; Evans, L. A.; Pond, S. M.; Cooper, D. A. Nat. Med. 2009, 15, 285. 24. Liu, J.; Lu, L. Angew. Chem., Int. Ed. 2007, 46, 7587.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
8–17 DNAzyme modified with purine analogs in its catalytic core: The conservation of the five-membered moieties of purine residues Wei Rong a, Liang Xu b, Yang Liu b, Jianping Yu a,⇑, Ying Zhou a, Keliang Liu b, Junlin He b,⇑ a b
College of Life Science, Guizhou University, Guiyang 550025, China Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
a r t i c l e
i n f o
Article history: Received 10 April 2012 Revised 8 May 2012 Accepted 9 May 2012 Available online 17 May 2012 Keywords: 8–17 DNAzyme Purine analogs Catalytic cleavage Chemical modification 7-Nitrogen atom
a b s t r a c t 8–17 DNAzyme is characterized by its recurrence in different in vitro selections and versatile cleavage sites, leading to extensive studies on its structural properties and applications. We evaluated the purine residues (A6, G7, G11, A12, G14, and A15) in the catalytic core of 8–17 DNAzyme of their five-membered ring moiety with purine analogs 1–5 to have an insight into the conservation of the residues at the level of functional groups. The 7-nitrogen atom in the AGC loop was demonstrated to be strictly conserved for the cleavage reaction. But such modifications exerted favorable effect at G11 of the base-pair stem and A12 in the single-strand loop, directing toward more efficient DNAzymes. Even the most conserved G14 could tolerate such modifications. These results demonstrated that chemical modification on the functional groups is a feasible approach to gain an insight into the structural requirement in the catalytic reaction of DNAzymes. It also provided modification sites for introduction of signaling molecules used for mechanistic and folding studies of 8–17 DNAzyme. Ó 2012 Elsevier Ltd. All rights reserved.
Since the appearance of small artificial DNAzymes 10–23 and 8–17 and the development of their wide-broad applications, many new DNAzyme entities have also been selected under specific conditions.1–4 Interestingly, numerous 8–17 DNAzyme variants (e.g., 17E and Mg5) have been selected independently from different in vitro selection experiments.5–9 Their catalytic cleavage activity and specific ion-dependence has led to a new class of metal biosensors.10–12 Such recurrence of 8–17 motifs and versatile cleavage sites13 initiated many studies on its catalytic mechanism and folding,14 either with residue replacement,15 spectroscopic methods,16,17 or contact photo-crosslinking experiments.18,19 In the catalytic core, all of them share a highly conserved AGC triloop, a Watson–Crick base-pair stem and the single-strand loop of four or five residues (Fig. 1).13 Each residue was supposed to offer base stacking and several functional groups to form a complex hydrogen-bond net responsible for the active conformation. Although we could not yet conclude with a precise catalytic site or a role of specific residues in the catalytic core of 8–17, extensive studies are still being conducted to gain an insight about its structure and mechanisms for a better design of metal biosensors and other applications. Considering that the residues at each position in the catalytic core is the result of repeated selection and the positive role of base stacking, we started the chemical modification on specific functional groups around nucleobases in the catalytic core, in ⇑ Corresponding authors. Tel.: +86 851 385 4677; fax: +86 851 385 6374 (J.Y.); tel./fax: +86 10 6821 1656 (J.H.). E-mail addresses: [email protected] (J. Yu), [email protected] (J. He). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.05.044
3'-TCC TAG AT rG rA CCG AGG TA-5' 2.1
5'-AGG ATC TA T 3
A
GGC TCC AT-3'
G 14 C C G AC G G 6A C G C 7
8-17 DNAzyme
8
Figure 1. The 8–17 DNAzyme designed against a DNA–RNA–DNA chimeric substrate. The arrow showed the cleavage site between diribonucleotide rA-rG. Bold letters indicated the most conserved purine residues.
order to have a better understanding of the conservation of nucleobases at the level of the functional groups and help the construction of more sensitive deoxyribozyme-based metal biosensors. Of the three structural domains in the catalytic core of 8–17 DNAzyme, there are eight purine residues, including three most conserved purine residues A6, G7, and G14, three guanine residues (G5, G10, and G11) in the base-pair stem, and A12 and A15 in the single-strand turn near the cleavage site. In this work, we present the evaluation of the five-membered ring moiety of these adenine and guanine residues except G5 and G10. The unnatural nucleoside analogs 1–520–22 (Fig. 2) with no changes on the Watson–Crick base-pairing functional groups were selected. By comparing with parent 8–17 DNAzyme, the contribution of 7-N atom as well as
4239
W. Rong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4238–4241
O
O
HN
HN N O
H2N HO
N
H2N HO
OH 1
HO
N
N O
HO
OH 4
N
OH 3 NH2
NH2
N
N N
N N O
HO
NH2
HN N O
N
N
OH 2
O
H2N
NH2
N N O
N
OH 5
Figure 2. Purine analogs used for the modification on purine residues in the catalytic core of 8–17 DNAzyme.
the five-membered ring moiety of purine residues was evaluated. Their phosphoramidites were prepared as previously reported (the preparation of phosphoramidite of 4 was presented in Supplementary data). 8–17 DNAzyme was designed against the sequence from the overlapping vpr and tat reading frames of HIV-1,23 and the target sequence was synthesized as a chimeric DNA–RNA–DNA, in which GA diribonucleotide was the cleavage site. 8–17 and its modified analogs were synthesized with conventional solid-phase synthesis and purified with denaturing PAGE (8% urea). All of them were characterized by MALDI-TOF (Table 1). Thermal measure-
Table 1 Characterization results of oligodeoxynucleotides by MALDI-TOF a
DNAzymes
Sequences
8-17DZ00
agg atc taT CCG AGC CGG ACG Agg ctc cat agg atc taT CCG A1C CGG ACG Agg ctc cat agg atc taT CCG A2C CGG ACG Agg ctc cat agg atc taT CCG 3GC CGG ACG Agg ctc cat agg atc taT CCG A4C CGG ACG Agg ctc cat agg atc taT CCG 5GC CGG ACG Agg ctc cat agg atc taT CCG AGC CG1 ACG Agg ctc cat agg atc taT CCG AGC CG2 ACG Agg ctc cat agg atc taT CCG AGC CG4 ACG Agg ctc cat agg atc taT CCG AGC CGG 3CG Agg ctc cat agg atc taT CCG AGC CGG 5CG Agg ctc cat agg atc taT CCG AGC CGG AC1 Agg ctc cat agg atc taT CCG AGC CGG AC2 Agg ctc cat agg atc taT CCG AGC CGG AC4 Agg ctc cat agg atc taT CCG AGC CGG ACG 3gg ctc cat agg atc taT CCG AGC CGG ACG 5gg ctc cat atg gag cca gta gat cct
8-17DZ01 8-17DZ02 8-17DZ03 8-17DZ04 8-17DZ05 8-17DZ06 8-17DZ07 8-17DZ08 8-17DZ09 8-17DZ10 8-17DZ11 8-17DZ12 8-17DZ13 8-17DZ14 8-17DZ15 D18
MS (calcd)
MS (found)
9212.4
9212.1
9211.4
9217.8
9212.4
9212.1
9212.4
9212.7
9269.2
9266.7
9269.6
9268.2
9211.4
9215.8
9212.4
9211.1
9269.6
9268.7
9212.4
9213.9
9269.6
9268.4
9211.4
9215.8
9212.4
9213.1
9269.6
9269.9
9212.4
9216.5
9269.6
9269.8
5521.0
5520.9
a The lowercase letters constitute both recognition arms, and the uppercase letters represent the catalytic core of 8–17 DNAzyme.
ment on the complexes between DNAzyme and its corresponding full-DNA substrate 50 -d(ATG GAG CCA GTA GAT CCT)-30 (D18, Table 1) in the reaction buffer indicated that all the modifications did not results in significant changes on thermal stabilities of the complexes (Supplementary data). The DNAzyme–substrate complex was formed in the reaction buffer. The cleavage activity of 8–17 and its analogs was determined under single-turnover conditions in the presence of 20 mM Mg2+, with DNAzymes of 100-fold more than the 32P-labeled substrate (Fig. 1). In addition to its repeated appearance in 8–17 variants, AGC loop has also been demonstrated to be highly conserved through mutagenesis with canonical and non-canonical residues, their functional groups, such as amino, keto, and 7-N atom are essential for the catalytic activity. In our evaluation system for modified 8– 17, as shown in Table 2, the same phenomenon was also observed.15 When the 7-N atom of G7 was deleted through the introduction of compound 1 at G7 in 8-17DZ01, a decreased kobs was obtained. Even a position shift of this nitrogen atom by compound 2 in 8-17DZ02 was not allowed, complete abolishment of cleavage activity was observed. Similar effect was produced when the 7-N atom of A6 was shifted to 8-position with compound 3 in 817DZ03. These observations indicated that A6 and G7 were probably located very close to the catalytic center, all functional groups around the two nucleobases might completely fold into the catalytic conformation, either by forming hydrogen-bonding network or positioning metal ions. In other words, the spacial distribution of these functional groups has been best defined by A6 and G7 as the repeated in vitro selection result. Further introduction of amino group at this loop with compound 4 or 5 led to new interactions accompanied by a complete loss of activity. This result further confirmed the absolute importance of the AGC loop in forming the catalytic conformation at the level of functional groups. The three base-pair stem has been supposed to play a structural role, offering stabilization for the catalytic conformation, based on the fact that G–C and A–T base pairs are well-tolerated, even mismatched base pairs were found in 8–17 variants. Especially, this stem could be replaced by T–Hg–T pair, with which mercuric ion switches on the stem formation and subsequently the cleavage reaction.24 Combining with fluorescent molecule labeling at the recognition arms, such 8–17 variants have been used as the biosensors for Hg2+. Based on these researches, the base pair in the stem was hypothesized to be located inside the helix, and their functional groups might not be involved in the catalytic conformation directly. Indeed, removal of 7-N atom by compound 1 at G11 did not produce significant effect on reaction rate (8-17DZ06, kobs = 0.0077 ± 0.0007 min1). However, when this 7-N was shifted to 8-position by compound 2 at G11, about twofold increase in reaction rate of 8-17DZ07 (kobs = 0.0131 ± 0.0005 min1) was produced. This slight improvement indicated that the five-membered
Table 2 kobs of 8–17 DNAzyme and its modified analogs measured under single-turnover conditionsa No.
kobs (min1)
No.
kobs (min1)
8-17DZ00 8-17DZ01 8-17DZ02 8-17DZ03 8-17DZ04 8-17DZ05 8-17DZ06 8-17DZ07
0.0064 ± 0.00055 0.0025 ± 0.00006 NDb ND ND ND 0.0077 ± 0.0007 0.0131 ± 0.0005
8-17DZ08 8-17DZ09 8-17DZ10 8-17DZ11 8-17DZ12 8-17DZ13 8-17DZ14 8-17DZ15
0.0103 ± 0.0010 0.0039 ± 0.00036 0.0101 ± 0.00105 0.0019 ± 0.00017 0.0098 ± 0.0013 0.0062 ± 0.0005 0.0014 ± 0.0001 <0.0001
a The cleavage reaction of DNAzyme (2 lM) on the 32P-labeled substrate (20 nM) were conducted in the buffer (50 mM Tris–HCl, 20 mM Mg2+) at 37 °C (more details see Supplementary data). b Not detected under present conditions.
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When G14 of 8–17 was further evaluated with compound 1 or 2, the kobs of new DNAzymes 8-17DZ11 (kobs = 0.0019 ± 0.00017 min1) and 8-17DZ12 (kobs = 0.0098 ± 0.0013 min1) demonstrated that 7-N and 8-N atoms at G14 were helpful for the cleavage reaction. Further perturbation with an extra amino group by compound 4 in this position could not offer more favorable effect on the reaction (8-17DZ13, kobs = 0.0062 ± 0.0005 min1). The cleavage pattern of these three DNAzymes within 300 min was shown in Figure 3B. This result told us that G14 is highly conserved as an integrate nucleobase, and its functional group distribution could be improved by functional group modification. For the adenine residues A12 and A15, mutations with compound 3 resulted in loss of activity, as shown in Table 2 (kobs = 0.0039 ± 0.00036 min1 for 8-17DZ09, and kobs = 0.0014 ± 0.0001 min1 for 8-17DZ14, respectively), it indicated that their 7-N atom was involved in the catalytic reaction. More adverse effect was observed for A15 when its 7-N was deleted. But the extra amino group by compound 5 in these two positions resulted in opposite effect. At A12, this extra amino group was beneficial for the reaction (8-17DZ10, kobs = 0.0101 ± 0.00105 min1), while it was deleterious for A15 (8-17DZ15). The cleavage behavior of these DNAzymes within 300 min was compared in Figure 3C. Based on the residue replacement and modification of functional groups on the catalytic core of 8–17 DNAzyme, we learnt that the residue conservation is determined by both base stacking and the interactions derived from functional groups. Chemical modifications on functional groups of specific nucleobases could give a more detailed picture of the residue involvement in the cleavage reaction. The two purine residues in the AGC loop was absolutely conserved at the level of functional groups, while the five-membered moiety of the conserved G14 could be modified with positive effect on the cleavage behavior. Based on the effect of compound 4 at G11 and A12, these two positions were the possible sites for introduction of signaling molecules used in the research of mechanism and folding of 8–17, through the 7-positioned amino group of compound 4. By this way, favorable modifications could be selected on the level of functional groups, either for more detailed understanding of the structural requirement in the catalytic reaction of DNAzymes, or for more efficient versions of DNAzymes. Acknowledgments This research was supported by National Natural Science Foundation of China 21072229 and Beijing Natural Science Foundation 7123223. Figure 3. The effect of purine analogs incorporated into 8–17 DNAzyme on the cleavage reactions: A, G11 replaced by 1 (8-17DZ06, d), 2 (8-17DZ07, N), 4 (817DZ08, ), parent 8–17 (j); B, G14 replaced with 1 (8-17DZ11, s), 2 (8-17DZ12, 4), 4 (8-17DZ13, }); C, A12 replaced by 3 (8-17DZ09,q) and 5 (8-17DZ10,w), A15 replaced by 3 (8-17DZ14,5), and 5 (8-17DZ15,.), respectively.
moiety of G11 might be involved in the cleavage reaction by its 7-N atom, and 8-N atom is a little more favorable for this positive effect than 7-N atom. Further introduction of compound 4 remained almost the same rate increase. Their cleavage pattern within 300 min was shown in Figure 3A. The effect of compound 4 at G11 and the extra amino group offers a potential spot for labeling 8–17 DNAzyme with signaling molecules in the studies of folding of 8–17 DNAzyme.17 The third domain in 8–17 DNAzyme is the single-strand turn close to the cleavage site, it differs from each other between its variants, ACGA in 8–17, TCGAA in 17E, and ACGAA in Mg5. C13G14 are the most conserved residues, any substitution with other three canonical residues led to deleterious effect on the cleavage activity.
Supplementary data Supplementary data (the preparation of phosphoramidite of compound 4 and Tm of the complexes between DNAzymes with the corresponding full-DNA substrate) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bmcl.2012.05.044. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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