Structural basis for recognition of Co 2+ by RNA aptamers Jan Wrzesinski and Stanisław K. Jo ´ z ´ wiakowski Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan ´ , Poland RNA molecules are involved in numerous fundamental cellular processes, such as replication, transcription and translation. Most recently, participation of small interfering RNA, microRNA and noncoding RNA in the regulation of gene expression and the development of variety organisms has been intensively investigated [1–3]. However, the interaction of RNA molecules with cellular components requires their proper folding into the active structure. This process is facilitated by the presence of cations, such as polyamines and mono- valent and divalent metal ions [4,5]. Determination of the precise location of the metal ions inside the RNA structure is important for a better understanding of RNA interactions with other components of the cell. Hence, many biophysical and biochemical methods have been developed for defining RNA ligands that coordinate metal ions. The most informative biophysical methods, which involve X-ray crystallography and NMR spectroscopy, provide details of the structure of the metal-ion bind- ing sites and their coordination spheres [6,7]. The disadvantages of X-ray crystallography and NMR spectroscopy include problems with respect to crystalli- zation and the need for isotope enrichment to resolve the RNA spectrum. Therefore, in order to gain a glo- bal insight into metal ion–RNA interactions, it is often necessary to conduct structural studies of metal ion binding modes in RNA molecules using biophysical methods simultaneously with other biochemical approaches. Metal ion-induced cleavage, an alternative approach of biochemical studies, is frequently used to identify those RNA stretches involved in the organization of metal ion binding site(s) in solution. This approach Keywords Co 2+ binding RNA aptamers; Co 2+ -induced conformational changes; kissing dimer; NAIM; oligomer hybridization ⁄ RNase H digestion Correspondence J. Wrzesinski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12 ⁄ 14, 61-704 Poznan ´ , Poland Fax: +48 61 8520532 Tel: +48 61 8528503 E-mail: wrzesinj@ibch.poznan.pl (Received 8 October 2007, revised 27 December 2007, accepted 5 February 2008) doi:10.1111/j.1742-4658.2008.06320.x Co 2+ binding RNA aptamers were chosen as research models to reveal the structural basis underlying the recognition of Co 2+ by RNA, with the application of two distinct methods. Using the nucleotide analog interfer- ence mapping assay, we found strong interference effects after incorpora- tion of the 7-deaza guanosine phosphorotioate analog into the RNA chain at equivalent positions G27 and G28 in aptamer no. 18 and G25 and G26 in aptamer no. 20. The results obtained by nucleotide analog interference mapping suggest that these guanine bases are crucial for the creation of Co 2+ binding sites and that they appear to be involved in the coordination of the ion to the exposed N7 atom of the tandem guanines. Additionally, most 7-deaza guanosine phosphorotioate and 7-deaza adenosine phos- phorotioate interferences were located in the common motifs: loop E-like in aptamer no. 18 and kissing dimer in aptamer no. 20. We also found that purine rich stretches containing guanines with the highest interference val- ues were the targets for hybridization of 6-mers, which are members of the semi-random oligodeoxyribonucleotide library in both aptamers. It tran- spired that DNA oligomer directed RNase H digestions are sensitive to Co 2+ and, at an elevated metal ion concentration, the hybridization of oligomers to aptamer targets is inhibited, probably due to higher stability and complexity of the RNA structure. Abbreviations c 7 AaS, 7-deaza adenosine phosphorotioate; c 7 GaS, 7-deaza guanosine phosphorotioate; NAIM, nucleotide analog interference mapping; NTA, nitrilotriacetate. FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS 1651 has been successfully used to detect various metal ions (e.g. Mg 2+ ,Pb 2+ ,Mn 2+ ,Eu 2+ ,Tb 3+ ,Co 2+ , etc.) in divergent RNA molecules involving tRNAs, ribo- zymes, and other RNA molecules [8–10]. The active involvement of metal ions in the cleavage of several ribozymes has been investigated using a ‘metal ion specificity switch assay’ with respect to sul- fur substitution of the oxygen atom in the phosphate group [11]. The cleavage efficiency for such modified ribozymes, strongly reduced in the presence of Mg 2+ , is rescued when thiophillic metal ions Mn 2+ ,Cd 2+ or Zn 2+ are added [12,13]. However, the precise determination of RNA ligands necessary for metal ion binding and RNA activities (mainly ribozymes) has become possible by applying nucleotide analog interference mapping (NAIM). In vitro transcription in the presence of 5¢-O-(1-thio)- nucleoside triphosphates enables incorporation of these modified nucleotides or nucleoside analogs with altered base moieties into the RNA chain, and I 2 cleavage allows determination of the atom in the bases that interferes with function [14,15]. NAIM has been used to identify the RNA ligands that interact with metal ions and the nucleotide modifications that are critical for retaining ribozyme activity [16–18]. In the present study, we applied the NAIM method to study the structural basis of the molecular mecha- nism underlying the binding of Co 2+ to RNA mole- cules using two aptamers, no. 18 and no. 20, which coordinate Co 2+ ions, previously selected in our labo- ratory, as research models [19]. Several purine N7 groups that interfere with Co 2+ were detected. Additionally, the influence of the Co 2+ binding on the aptamer structure using hybridization of a 6-mers semi-random oligodeoxynucleotide library and RNase H digestion was investigated. Results and Discussion Synthesis of phosphorothioate nucleoside modified RNA aptamers As prepared using the T7 transcription system, a pool of aptamers (Fig. 1) carrying randomly distributed phosphorothioate modifications, as well as 7-deaza guanine or adenine analog substitutions, was analyzed to determine which RNA ligands interfere with Co 2+ binding. It is worth noting that T7 RNA polymerase only incorporates the S P -stereoisomer of phosphoro- thioate modified nucleotides into the RNA chain and causes inversion of the configuration at the a-phospho- rus atom, resulting in R P -phosphorothioate substitu- tion [16]. We used nitrilotriacetate (NTA) resin with immobilized Co 2+ to separate the partially modified aptamers into Co 2+ binding and nonbinding fractions by the metal ion affinity chromatography approach and fractions were then subjected to iodine cleavage. A comparison of cleavage patterns of both fractions enables determination of which RNA ligands actively participate in the Co 2+ binding event. Effect of R P -phosphorothioate nucleoside modification on Co 2+ binding Only three phosphorothioate interferences within the loop region of both aptamers were observed (Figs 2– 4). Two weak UaS(j ¼ 1.8) interferences took place at U42 in aptamer no. 18 and at U44 in aptamer no. 20 (in equal positions; the second nucleotide at the 3¢-end of the loop; Fig. 4). The third CaS moderate interference (j ¼ 2.4) occurred at C36 in aptamer no. 18. Generally, relatively low interference effects were discovered after replacing the nonbridging pro-R P oxy- gen atom with sulfur. The reason for the above obser- vation might be the dominant contribution of base Fig. 1. The secondary structure of the in vitro selected aptamers that bind Co 2+ . The locations of Co 2+ -induced cleavage sites are shown by open arrows. Recognition of Co 2+ by RNA aptamers J. Wrzesinski and S. K. Jo ´ z ´ wiakowski 1652 FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS moieties in the formation of the Co 2+ binding site architecture in the aptamers. Therefore, replacement of the nonbridging oxygen atom with sulfur in the phos- phate group had a small effect on Co 2+ binding in contrast to ‘soft’ thiophillic metal ions Cd 2+ ,Mn 2+ and Zn 2+ , which discriminated between the oxygen and sulfur atoms, with the latter being preferentially bound [20]. Importance of the N7 group of purines for the development of Co 2+ binding sites In aptamer no. 18, we found weak 7-deaza adenosine phosphorotioate (c 7 AaS) interferences at A26 (j = 1.9) and A41 (j = 1.4) (Figs 2 and 4). By con- trast, for 7-deaza guanosine phosphorotioate (c 7 GaS), five interferences were observed and three of them were localized at the 5¢-side of the loop. The strongest interferences (j = 4.0 and 3.6) were detected at G27 and G28, respectively. Interference at G25 was less prominent (j = 1.9). Additionally, there were two weak interferences at G37 (j = 2.0) and G38 (j = 1.9), within the previously identified Co 2+ - induced cleavage sites [19]. The strongest interferences occurred at G27 and G28, which are positioned within the purine rich stretch 19-AGGCGAGAGG-28. It is known that such regions containing purine stretches are often involved in strong stacking interactions, reducing their flexibility [21,22]. Thus, positioned within the stacked, more rigid 19-AGGCGAGAGG-28 stretch, guanine bases would be accessible to interact with the Co 2+ ion immobilized on NTA resin, and the ion is probably coordinated to N7 atom of the imidaz- ole ring of guanines. Yet the possibility that deletion of the N7 group of purines may result in an alternative structure of the aptamer cannot be excluded. However, Fig. 2. Iodine cleavage analysis of phospho- tioate nucleoside analogs modified 5¢- 32 P labeled Co 2+ binding aptamer no. 18. Lanes: C, reaction control of the F N fraction; F N , RNA fraction which is not bound to Co 2 - NTA resin; F B , RNA fraction that is effec- tively bound to Co 2+ -NTA resin and is eluted with 2 m M concentration of Co 2+ ; L, form- amide ladder; T1, limited hydrolysis by RNase T 1 . Guanine residues are labeled on the right. Sites of interference are denoted with arrows. J. Wrzesinski and S. K. Jo ´ z ´ wiakowski Recognition of Co 2+ by RNA aptamers FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS 1653 these purines are located in the single-stranded region accessible for chemical probing [19]. Thus, N7 atoms of the above guanine residues are not involved in inter- actions with other base ligands or in aptamer structure development. Interestingly, similar binding properties of Co 2+ to the N7 atom of guanine residues were previously observed in the crystal structure of the orthorombic form of yeast tRNA Phe [23]. In the predicted loop E-like motif, bases G25 ⁄ A43, as well as G27 ⁄ A41, form a sheared G-A base pairing, whereas the A26-U42 base pair displays a reversed Hoogsteen geometry [24] (Fig. 5). Thus, c 7 AaS interfer- ences at A26 and A41 confirm that the N7 atom of these adenine bases is involved in the formation of non- standard H-bonding. Additionally, in the proposed loop E-like motif structure, the aforementioned N7 position of G25 is exposed to interaction with Co 2+ and such interactions may stabilize this motif. Furthermore, the N7 atom of G37 and G38 partici- pates in Co 2+ coordination or is involved in other interactions that build Co 2+ binding sites. However, the interference j values are at least two-fold lower in comparison with G27 and G28, indicating that those interactions are weaker. Unlike in aptamer no. 18, the c 7 AaS interferences were not observed in aptamer no. 20 (Figs 3 and 4). In the case of the c 7 GaS modified aptamer, four inter- ferences were found. Strong interferences at G25 (j = 3.7) and G26 (j = 3.5) are located at the same positions as the interferences at G27 and G28 in apt- amer no. 18 (i.e. in the third and fourth positions at the 5¢-end of loop, within 19AGGCGA GG-26, a pur- ine stretch two nucleotides shorter than in the case of aptamer no. 18). The determined j values were very similar: 4.0 and 3.6 for G27 and G28 in aptamer no. 18 and 3.7 and 3.5 for G25 and G26 in aptamer Fig. 3. Iodine cleavage analysis of phospho- tioate nucleoside analogs modified 5¢- 32 P labeled Co 2+ binding aptamer no. 20. Lanes: C, reaction control of the F N fraction; F N , RNA fraction which is not bound to Co 2 - NTA resin; F B , RNA fraction that is effec- tively bound to Co 2+ -NTA resin and is eluted with a 2 m M concentration of Co 2+ ; L, formamide ladder; T1, limited hydrolysis by RNase T 1 . Guanine residues are labeled on the right. Sites of interference are denoted with arrows. Recognition of Co 2+ by RNA aptamers J. Wrzesinski and S. K. Jo ´ z ´ wiakowski 1654 FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS no. 20, respectively. Additional weak interferences (j < 2) at G39 and G40 were discovered. According to our earlier assumption, in aptamer no. 20, the self- complementary region 35-ACGCGG-40 was predicted to be involved in the formation of the kissing dimer [19]. The appearance of this effect in aptamer no. 20 was confirmed experimentally (Fig. 6). In the presence of a 1 mm concentration of Co 2+ , as well as Mg 2+ ions, mobility shift on the nondenaturing gel was observed, indicating that divalent metal ions Co 2+ and Mg 2+ are necessary for kissing dimer formation in a nonspecific manner. We believe that interferences within the self-complementary region of aptamer no. 20 indicate the additional stabilization of the kiss- ing complex by interaction of Co 2+ with the N7 atom of guanine bases, namely nonstandard G–A and the neighboring G–C base pairs. Interestingly, analysis of the crystal structure of RNA fragments mimicking the HIV-1 virus subtype A kissing complex, crystallized in the presence of different metals, revealed that Mg 2+ , Fig. 4. Summary of the interference effects within the Co 2+ bind- ing aptamer no. 18 and no. 20 defined by NAIM. The histogram represents the secondary structure of the aptamer loop regions. The bars are correlated with the determined magnitude of interfer- ence (j values). Analogs used in NAIM are marked with appropriate colors. Dotted lines in aptamer no. 18 mark the loop E-like base pairing, and the solid line in aptamer no. 20 indicates the nucleo- tides involved in the dimer complex. A B C Fig. 5. The aptamer no. 18 loop E-like motif structure: (A) nucleo- tides involved in loop E motif formation; (B) hydrogen bonding pat- terns in the extended G-A; and (C) reversed Hoogsteen A-U nonstandard base pairs. 1 mM EDTA Aptamer no. 18 Aptamer no. 20 dime r H 2 O + ++ ++ ++ +– – – ––– –– – –– –– –––– – – ––––– 1 m M MgCl 2 1 mM CoCl 2 Fig. 6. (A) Gel shift assay dimer formation by the aptamers no. 18 and no. 20. 5¢- 32 P labelled RNA samples containing 20 mM Tris–HCl pH 7.5, 40 m M NaCl dimerization buffer were supplemented with 1m M concentration of EDTA, Mg 2+ and Co 2+ , respectively. (B) Scheme of proposed secondary structure of aptamer no. 20 dimer complex. The self-complementary sequence ACGCGG is shown. J. Wrzesinski and S. K. Jo ´ z ´ wiakowski Recognition of Co 2+ by RNA aptamers FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS 1655 as well as Co 2+ ,Zn 2+ and Mn 2+ , preferentially bind to the N7 atom of guanine bases in such a motif [25]. Recognition of Co 2+ by RNA aptamers The data presented here reveal the importance of the N7 atom of purines in the organization of the Co 2 binding site in selected aptamers and their involvement in the metal ion coordination (Fig. 4). Participation of the N7 atom of purines in the organization of the ter- tiary structure of other RNAs has been extensively investigated by the NAIM method. Heide et al. [26] have demonstrated the significance of this position in purines upon binding of tRNA to Escherichia coli RNase P. The N7 position of the adenines is also nec- essary for the catalytic activity of the RNase P–sub- strate conjugate [27]. Using the NAIM assay, six adenines critical for self-cleavage have been identified. The application of a set of phosphorothioate nucleo- side analogs, including 7-deaza-purine analogs, to examine the interactions of low molecular ligand glu- cosamine 6-phosphate with glmS ribozyme in the pres- ence of Mg 2+ demonstrated the importance of the N7 group of purines and Mg 2+ ions in the organization of the catalytic site of the ribozyme and in the glucosa- mine 6-phosphate recognition process [28]. We show that, in the selected aptamers, the N7 position of some guanine bases is needed for binding of Co 2+ immobi- lized on the NTA resin. As Co 2+ ions usually contain six coordination sites and four of them are occupied upon complexation with the resin, only two sites remain available for interactions with RNA ligands, including the N7 atom of purines. Therefore, there is the possible involvement of the N7 atom of the neigh- boring tandem guanines, G27 and G28 or G37 and G38 in aptamer no. 18, as well as G25 and G26 or G39 and G40 in aptamer no. 20, in Co 2+ binding. Interestingly, a similar metal ion binding mode to the tandem guanines has been observed in the resolved crystal structure of leadzyme [29]. Strontium ions, which mimic lead ions, have been bound directly or water mediated to the N7 position of several purines, mainly guanine tandems. In other RNAs whose struc- tures have been determined with atomic resolution, such as hairpin ribozyme and the P4–P6 domain of the group I intron, binding of metal ions in a manner identical to that of guanine tandems has been identi- fied [30,31]. The previously performed Co 2+ -induced cleavage of aptamers revealed a doublet of scissions occurring at nucleotides G37 and G38 in aptamer no. 18 and nucleotides A31 and G32 in aptamer no. 20 [19]. Additionally, the determined cleavage rate constant for aptamer no. 18 was three-fold higher than that for apt- amer no. 20. It is well established that the rate of metal ion-induced cleavage strongly depends on the distance between the ion in its strong binding site and the 2¢OH group of ribose moiety involved in the scission phosphodiester bond mechanism [10]. Thus, R P -phosphorothioate CaS interference at C36 in apt- amer no. 18, adjacent to Co 2+ -induced cleavage sites, strongly suggests that this region is involved in direct metal ion–RNA interactions. In the case of aptamer no. 20, those interactions are presumably weaker; hence, interference was not observed. Influence of Co 2+ on aptamer structures in solution To gain a better insight into the effect of Co 2+ binding on the RNA structure, we applied a semi-random DNA library of 6-mers and RNase H, an endonucleolytic ribonuclease that specifically recognizes the DNA–RNA duplex and digests it. It has been shown that hybridiza- tion of short oligodeoxyribonucleotides to RNAs is strongly affected by RNA target structures, which results in changing RNase H digestion efficiency [32,33]. One big advantage of this approach involving the appli- cation of a semi-random library is that no knowledge of the RNA structure is required to determine the DNA oligomer sequence that effectively hybridizes to the RNA target. We applied a semi-random library contain- ing a single fixed nucleotide (A,G, C or T), located in the third position of the oligodeoxyribonucleotide chain and five random nucleotides; thus, the library consisted of 4096 members (Fig. 7A). Knowing the RNase H digestion preferences that cleave RNA at the end of bound DNA oligomer, it is possible to correlate the RNase H digestion sites with the most likely positions of hybridized DNA 6-mers [32,33]. In a first step, we determined the RNase H digestion sites and the possible location of the binding region within the aptamer structures to which 6-mers, mem- bers of the oligodeoxyribonucleotide library, hybridize. In aptamer no. 18, digestions took place at G27, G28, G30 and G31 at the 5¢-end of the loop (Fig. 7B,C). Additionally, a doublet at G35 and C36 was identified. However, in aptamer no. 20, four digestion sites were found: G26, U27, A30 and A31 at the 5¢-end of loop. In both loops, 6-mer oligomers hybridize to the 5¢-end of the loops and propagate to nucleotides involved in the formation of the helix. The strong preference for the 5 ¢-side of the loops in comparison with the 3 ¢ -side may be explained by different sequences of both sides. As noted above, both aptamers contain purine rich stretches that could be involved in stacking Recognition of Co 2+ by RNA aptamers J. Wrzesinski and S. K. Jo ´ z ´ wiakowski 1656 FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS interactions. Such specific characteristics of RNA, involving the U turn, the formation of stable tertiary base pairs and, particularly, the stacking interactions that ensure the helical order of single-stranded regions, are the main factors determining the efficiency of hybridization of short DNA oligomers to the RNA target [32,33]. Protection of the 35-ACG-37 region in aptamer no. 20 against hybridization of the 6-mers oli- godeoxyribonucleotide library is due to the formation of the kissing loop complex (Fig. 6). Additionally, the observations of RNase H specificity are well correlated with the results obtained by NAIM noted earlier. The main c 7 GaS interference sites are positioned in the same regions: 19-AGGCGAGAGG-28 at G27 and A B C Fig. 7. (A) Sequence of the oligodeoxyribo- nucleotide library used. (B) Autoradiograms of RNA fragments showing digestion of Co 2+ binding aptamers with RNase H in the presence of semi-random libraries. 5¢- 32 P end-labeled RNAs were used and the reac- tion products were analyzed on the gel. Lanes: –, reaction control; a, g, c, t, parts of semi-random library; L, formamide ladder; T1, limited hydrolysis by RNase T 1 . Guanine residues are labeled on the right. (C) Loca- tion of RNase H digestion sites displayed on the aptamer secondary structure models. Gray lines along the aptamer sequences show the possible position of 6-mer oligode- oxyribonucleotides hybridizing to the RNA targets. Letters with arrow denote diges- tions to which the corresponding 6-mer oli- godeoxyribonucleotides could be assigned. J. Wrzesinski and S. K. Jo ´ z ´ wiakowski Recognition of Co 2+ by RNA aptamers FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS 1657 G28 in aptamer no. 18 and 19-AGGCGAGG-26 at G25 and G26 in aptamer no. 20, which we postulate to be involved in Co 2+ coordination. The dependence of oligomer hybridization and RNase H digestion of aptamers on Co 2+ concentration was studied with a library (i.e. the part of the semi- random library that contains adenosine in the third position) because this part of the library revealed the most prominent and specific RNase H digestion sites (Fig. 7B,C). We detected inhibition of RNase H diges- tion efficiency with an increased Co 2+ concentration despite the presence of Mg 2+ at a 10 mm concentra- tion, as necessary for enzyme activity (Fig. 8). The inhibition constants, determined as the concentration of Co 2+ at which the extent of RNase H digestion was reduced by half, were approximately 0.5 mm for both aptamers. Subsequently, to exclude the possibility that Co 2+ would inhibit RNase H activity at a higher con- centration, we used another RNA model, antigenomic delta ribozyme, which has been well characterized in our laboratory [34]. Previously, we mapped the hybrid- ization sites of the 6-mer oligodeoxyribonucleotide library in delta ribozyme using an RNase H digestion assay and they have appeared to be localized within the single-stranded P1 and J1 ⁄ 4 regions [32]. Delta ribozyme is highly active in the presence of a 1 mm concentration of Co 2+ ions; therefore, these ion bind- ing sites probably occur inside the ribozyme structure [35]. Strikingly, we did not observe reduction of the RNase H digestion efficiency, but an increase of the digestion yield when Co 2+ was added, even at a 3 mm concentration. We assume that the lower RNase H digestion efficiency of the studied RNA aptamers is mainly related to the stabilization of their structures in the presence of Co 2+ ions. Presumably, the regions to which DNA oligomers hybridize become less accessible in more compact aptamer structures upon Co 2+ bind- ing and this process prevents the DNA–RNA duplex formation necessary for the RNase H digestion event. A reverse effect takes place in the antigenomic delta ribozyme. The presence of Co 2+ presumably desta- bilizes or rearranges the ribozyme structure facilitating hybridization of DNA oligomers because an increase of RNase H digestion efficiency was observed. The influence of Co 2+ ions on the global structure of aptamers has been studied by applying the UV melting technique (data not shown). We observed an increase of the melting temperature from 69.7 °C to 71.8 °C for aptamer no. 18, and from 64.9 °C to 66.2 °C for aptamer no. 20, despite the low concentration of Co 2+ 0 0.01 0.06 Aptamer no. 18 Aptamer no. 20 Antigenomic delta ribozyme 0.1 0.2 0.3 0.5 1 2 30.03 L T1 G38 G35 G31 C 0 0.01 0.06 0.1 0.2 0.3 0.5 1 2 30.03 L T1 C 0 0.1 0.2 0.3 0.5 1 2 30.05 L T1 CC1 Co [mM] 2+ G32 G37 G29 G26 G42 G35 0.0 1.0 2.0 3.00.5 1.5 2.5 Co 2+ concentration [mM] 0.0 1.0 2.0 3.00.5 1.5 2.5 Co 2+ concentration [mM] 0.0 1.0 2.0 3.00.5 1.5 2.5 Co 2+ concentration [mM] 0.0 0.2 0.4 Fraction cleaved 0.6 0.8 1.0 0.0 0.2 0.4 Fraction cleaved 0.6 0.8 1.0 0.0 0.2 0.4 Fraction cleaved 0.6 0.8 1.0 A B Fig. 8. (A) Digestion of Co 2+ binding aptamers and antigenomic delta ribozyme with RNase H in the presence of a-library (ie: the part of the semi-random library that contains adenosine in the third position) at different Co 2+ concentrations. Figure labelling is the same as in Fig. 7. C, incubation control; C1, incubation control in the presence of a 3 m M Co 2+ concentration. (B) Graphical representation of the dependencies of RNase H digestion efficiency on Co 2+ concentration of aptamers no. 18 and no. 20 and antigenomic delta ribozyme. Recognition of Co 2+ by RNA aptamers J. Wrzesinski and S. K. Jo ´ z ´ wiakowski 1658 FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS (0.1 mm). At a higher Co 2+ concentration, significant degradation of RNA during UV melting experiments was detected. This observation supports the above suggestion, as formulated on the basis of oligomer hybridization and RNase H digestion, that the binding of Co 2+ to in vitro selected Co 2+ -specific aptamers stabilizes their structure, even at a low metal ion concentration. In the present study, we postulate that Co 2+ binding to the aptamer structures induces the increase in their stabilities. A similar situation has been observed for riboswitches, which are highly structured domains that reside in the 5¢-UTR region of mRNA and affect gene expression [36]. The binding of some low molecular ligands (e.g. FMN, adenosylocobalamin, guanine, l-lysine) to the ‘aptamer domain’ of riboswitches stabi- lizes the specific RNA structure involved in the regula- tion of gene expression at a transcription or translation level. It is also known that attachment of the Co 2+ binding domains to the allosteric hammerhead ribo- zyme may regulate its catalytic activity [37]. More recently, riboswitches that respond to the cellular con- centration of Mg 2+ by forming a stable hairpin that affects the transcription level of Mg 2+ transporter gene expression (MgtA and MgtE in bacteria Salmonella enterica and Bacillus subtilis, respectively) have been described [38,39]. The above information, together with the data presented here, indicates that possibly also Co 2+ binding RNA molecules responding to the Co 2+ concentration may regulate RNA activity. Conclusions The significance of Mg 2+ for RNA stability, RNA catalysis and the regulation of gene expression is well established. However, information concerning partici- pation of other metal ions with distinct chemical prop- erties (e.g. Co 2+ , a member of the transition metal ion group), in such processes, and particularly the molecu- lar recognition of Co 2+ by RNA, still remains limited and requires further study. The application of phosphorothioate 7-deaza-purine analogs and the NAIM approach for studying the structure of Co 2+ binding sites in aptamers has revealed the importance of the N7 group of guanine bases. In both aptamers, we identified several tandems of guanines involved in Co 2+ binding or the develop- ment of aptamer structures. Additionally, other ele- ments of the secondary structure of aptamers, such as the E-like loop and kissing complex motifs, appear to be important in the formation of the architecture of Co 2+ binding sites. However, in the case of aptamer no. 20, which contains the self-complementary 35-AC GCGG-40 sequence, we have confirmed the appear- ance of a kissing loop complex motif in the presence of divalents Co 2+ or Mg 2+ . This observation is in line with NAIM results showing that the N7 groups of G39 and G40 interfere with Co 2+ . We would like to emphasize the importance of the purine rich stretches with stacking interactions for the binding of Co 2+ and other nucleic acid molecules. Regions 19-AGGCGAGAGG-28 in aptamer no. 18 and 19-AGGCGAGG-26 in aptamer no. 20 contain a tandem of guanines with the highest c 7 GaS interfer- ence values, thus indicating direct coordination of Co 2+ . The same regions are targets for 6-mer oligode- oxyribonucleotides, as confirmed in the present study using semi-random DNA library hybridization and an RNase H digestion assay. Moreover, the binding of Co 2+ to aptamers induces conformational changes that result in the stabilization of the RNA structures, which was confirmed in two independent experiments, namely (a) the hybridization of a semi-random library and RNase H digestion and (b) temperature-dependent UV melting. Experimental procedures Materials NTA resin was obtained from Novagen (Darmstadt, Ger- many); all chemicals were obtained from Serva (Heidelberg, Germany) or Fluka (Buchs, Switzerland). Phosphorothioate nucleotides (NTPaS) and c 7 AaS were purchased from Glen Research (Starling, VA, USA), except for c 7 GaS, which was purchased from IBA (Berlin, Germany). Enzymes: T7 RNA polymerase and DNA Taq polymerase T4 polynucleo- tide kinase were obtained from MBI Fermentas (Vilnius, Lithuania). [c- 32 P]ATP (5000 CiÆmmol )1 ) was obtained from Hartmann Analytic (Braunschweig, Germany). DNA template construction The following oligodeoxynucleotides were used for con- struction of the DNA templates: LM47: 5¢-GCGAGCTCT AATACGACTCACTAT GGGCATA nCGTTAGGCTGTA GGC-3¢, LM18: 5¢-CGAAGCTTGCATATGCTACGCT GAGGCGATAT TTCC GCT TTCC TCTC GCCTACAGCC TAACGTATGCCC-3¢ and LM20: 5¢-CGAAGCTTGCA TATGCTACGCTGAGGCUATTACCGCGTTTCTTCCA CCTC GCCTACAGCCTAACGTATGCCC-3¢ (letters in italic indicate the T7 RNA promotor, complementary sequences are underlined). Oligomers were deprotected after synthesis and purified on denaturing 8% (w ⁄ v) polyacryl- amide gel. Equimolar amounts of oligomers LM 47 and LM 18 or LM 20 were annealed and double-stranded DNA J. Wrzesinski and S. K. Jo ´ z ´ wiakowski Recognition of Co 2+ by RNA aptamers FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS 1659 template was generated by PCR. The reaction mixture con- tained 1.5 lm of both LM 47 oligomers and 0.3 lm of LM 18 or LM20, 10 mm Tris–HCl, pH 7.0, 2 mm MgCl 2 , 150 mm KCl, 0.1% Triton X-100, 200 lm each of dNTP and 60 UÆmL )1 of DNA Taq polymerase. The reaction was performed on Biometra (Go ¨ ettingen, Germany) UNO II thermocycler for six cycles (30 s at 93 °C, 30 s at 55 °C and 1 min at 72 °C). The double-stranded DNA was extracted with phenol ⁄ chloroform (1 : 1), precipitated with buffer: 3 m sodium acetate, pH 7.0 ⁄ ethanol (1 : 9) at )20 °C over- night. The dsDNA template was recovered by centrifuga- tion, dissolved in TE buffer and used in the transcription reaction. RNA preparation The modified RNA aptamers were prepared by the in vitro transcription reaction where the typical transcription reac- tion contained 0.5 lm dsDNA template, 40 mm Tris–HCl, pH 7.0, 10 mm MgCl 2 , Triton X-100, 2 mm spermidine, 5mm dithiothreitol, 1 mm each NTP, and 2000 UÆmL )1 T7 RNA polymerase. Additionally, the reaction mixtures con- tained one from the following phosphorothioate analogs: 0.05 mm ATPaS, 0.07 mm UTPaS, 0.07 mm CTPaS, 0.1 mm GTPaS, as well as 0.05 mm c 7 ATPaS, 0.1 mm c 7 GTPaS. To facilitate 5¢ labeling, 4 mm guanosine was added. Following incubation of the mixture at 37 °C for 4 h and purification on polyacrylamide gel, the RNA tran- scripts were excised, eluted, precipitated with ethanol, and RNA was recovered by centrifugation and dissolved in 10 mm Tris–HCl, pH 7.0, 0.1 mm EDTA. Nucleotide analogs interference mapping Prior to the interference procedure, RNA transcripts were 5¢-end labeled with [c- 32 P]ATP and polynucleotide kinase under standard conditions. Labeled aptamers (typically 2–3 · 10 6 c.p.m., 20 pmol of RNA supplemented with unlabeled RNA to a final 0.2 lm concentration) were sub- jected to a denaturation–renaturation procedure in 200 lL of the standard binding buffer A (40 mm Hepes, pH 8.0, 400 mm NaCl, 1 mm MgCl 2 ); RNA was incubated at 70 °C for 5 min, and slowly cooled to 25 °C. The aptamers were bound to Co 2+ -NTA resin, mixed gently with resin slurry for 5 min and, subsequently, the resin was washed with six volumes (400 lL each) of buffer A to remove the unbound RNA. In the next step, the resin was washed with addi- tional six volumes of buffer A containing 2 mm CoCl 2 .To both fractions, 1 ⁄ 10 volume of sodium acetate (pH 7.0) and 3 volumes of ethanol were added and the mixtures were stored at )20 °C overnight. The RNA was recovered by centrifugation and dissolved in water. The RNA fraction containing molecules that specifically bind to Co 2 -NTA resin (F B ) and the fraction deprived of such properties (F N ) were subjected to cleavage of phosphorothioate linkage in the presence of 10 mgÆmL )1 iodine ⁄ ethanol solution. The cleavage was carried out at 37 °C for 15 min; subsequently, the RNA was precipitated, recovered by centrifugation and dissolved in loading buffer (7 m urea ⁄ dyes 10 mm EDTA). Patterns of I 2 cleavage were compared on 8 m urea ⁄ 12% polyacrylamide gel alongside alkaline hydrolysis ladders and partial digestion with RNase T 1 . Products were visualized by autoradiography or quantified using a PhosphoImager Typhoon 8600 (Uppsala, Sweden) with imagequant software (Uppsala, Sweden). The interference j value was calculated from the equation: j ¼ band intensity at nucleotide x ðÞ P band intensity at nucleotide aÀz ðÞ NTP (band intensity at nucleotide xÞ P band intensity at nucleotide aÀz ðÞ NTPaS Interferences were considered as: weak, j = 1.5–2.0; mod- erate, 2.0–2.5; and strong, > 2.5. RNase H RNA mapping experiment The RNA aptamers were prepared by in vitro transcription, under the conditions described above, using unmodified NTPs, purified and labeled at their 5¢-end. Prior to digestion with E. coli, RNase H 5¢- 32 P labeled RNA was renatured in buffer (40 mm Tris–HCl, pH 8.0, 40 mm KCl, 10 mm MgCl 2 , 1mm dithiothreitol and 0.1 mm EDTA). Subsequently, RNase H was added to a final concentration of 250 UÆmL )1 . The digestion reactions were initiated by adding separately four parts of the DNA 6-mers library with the appropriate fixed nucleotide to four RNA target samples to a final con- centration of 200 lm (i.e. 5000-fold excess over the RNA concentration). The mixtures were incubated at 37 °C for 30 min, quenched with an equal volume of 7 m urea ⁄ 20 mm EDTA and immediately frozen on dry ice. In vitro RNA aptamer dimerization assay Aliquots of 5¢ labeled RNA aptamers, supplemented with unlabeled RNA to a concentration of 250 nm, were heated at 100 °C for 1 min and cooled on ice for 10 min. Then dimerization buffer containing, respectively, 20 mm Tris- HCl, pH 7.5, 40 mm NaCl alone, or supplemented with 1mm EDTA, MgCl 2 and CoCl 2 , was added. Subsequently, the reaction mixture was heated for 10 min at 25 °C. After adding 1 ⁄ 5 volume 30% glycerol, the samples were loaded directly on the 12% nondenaturing polyacrylamide gel. The electrophoresis under nondenaturing conditions was carried out at room temperature using 20 mm Tris–HCl, pH 7.5, 40 mm NaCl, 1 mm MgCl 2 electrophoresis buffer. Acknowledgements We are very grateful to Professor Jerzy Ciesiolka for critically reading the manuscript and for helpful Recognition of Co 2+ by RNA aptamers J. Wrzesinski and S. K. Jo ´ z ´ wiakowski 1660 FEBS Journal 275 (2008) 1651–1662 ª 2008 The Authors Journal compilation ª 2008 FEBS [...].. .Recognition of Co2+ by RNA aptamers ´´ J Wrzesinski and S K Jozwiakowski discussions throughout the course of this work We ´ wish to thank Ms Barbara Smolska for her excellent technical assistance We also thank the reviewers for their comments on the manuscript This work was supported by grant 6 P04A 081 21, from the Polish Committee for Scientific Research as well as the Bioorganic Chemistry and Structural. .. 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