Studies on Tris (2-aminobenzimidazole)-PNA Based Artificial

Article pubs.acs.org/bc

Studies on Tris(2-aminobenzimidazole)-PNA Based Artificial Nucleases: A Comparison of Two Analytical Techniques Plamena Dogandzhiyski,†,§ Alice Ghidini,‡,§ Friederike Danneberg,† Roger Strömberg,‡ and Michael W. Göbel*,† †

Institute for Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Max-von-Laue-Str. 7, D-60439 Frankfurt am Main, Germany ‡ Department of Biosciences and Nutrition, Karolinska Institutet, Novum, S-14157, Huddinge, Sweden S Supporting Information *

ABSTRACT: A new peptide nucleic acid (PNA) construct carrying a tris(2-aminobenzimidazole) phosphodiester cleaver is presented. This non-metal-based artificial nuclease hydrolyzes RNA substrates that form a bulge upon binding to the PNA. Reaction rates depend on the bulge sequence. For conjugates of tris(2-aminobenzimidazole), substrate turnover is shown for the first time. Two methods of analysis for the kinetics are compared: IE-HPLC separation of oligonucleotide fragments and analysis of Cy5-labeled oligonucleotide fragments by denaturating PAGE on a DNA sequencer, respectively. The different methods give rates that are in the same range where, in general, the substrates for the sequencer method give slightly lower rates.

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(PNA) based conjugate carrying a Cu2+-2,9-dimethyl-phenanthroline cleaving unit. It attains substrate half-lives as short as about 30 min.21 In that work, reaction kinetics and product identification was based on HPLC separation of fragments and mass spectrometric analysis. Metal-free synthetic nucleases, on the other hand, are not at risk to get deactivated and to cause toxic effects by the loss of metal ions.22−26 However, at present they are considerably less efficient in cleaving RNA as compared to the best metal-ion systems. Oligonucleotide conjugates of tris(2-aminobenzimidazole) cleave their RNA targets with half-lives ranging from 11 to 20 h.27,28 These studies were based on a completely different analytical approach and the question arises to what extent the results depend on the chosen method. Our two laboratories therefore decided to try a cross-check of analytical methods to identify their individual strengths which may also be advisory to other practitioners in the field. In addition, we found that aminobenzimidazole catalysts, when fixed opposite an RNA bulge, exhibit multiple substrate turnover as shown before for metal-ion systems. A third result found with aminobenzimidazole conjugates was a distinct dependence of reaction rate on bulge sequences, albeit not as pronounced as in the Cu2+phenanthroline system.

INTRODUCTION Phosphoric acid diesters, forming anions at neutral pH, are perfectly protected against hydrolysis by their negative charge. Half-lives of typical phosphodiester linkages in DNA, therefore, are in the range of 100 000 years.1 In contrast, RNA is much more susceptible to hydrolysis. The normal mechanism of RNA cleavage involves intramolecular attack of the 2′ OH group at phosphorus, a reaction largely accelerated by the neighboring group effect. The stereoelectronic requirement of this mechanism is a colinear arrangement of nucleophile, phosphorus, and leaving group. In regular duplex structures, large deviations from the optimal 180° angle prevent RNA cleavage by the in-line mechanism. Bulges, loops, and unstructured RNA parts, due to their conformational flexibility, may fulfill the stereoelectronic requirement and thus are prone to cleavage.2−7 In special cases, phosphodiesters may even become preorganized in the in-line conformation and exhibit accelerated cleavage. This kind of conformational control is an important factor to explain, for example, the efficiency of hammerhead ribozymes.8 The design of artificial ribonucleases which induce site and substrate specific cleavage is of significant research interest because of their potential application in RNA therapeutics. RNA cleavers which contain metal ions show promising results and they can be used as tools in molecular biology.9−11 By conjugation with oligonucleotides, specific cleavage sites within the RNA target can be addressed.12−17 To obtain catalytic turnover of substrate and faster cleavage rates, different bulge forms have been examined.18−20 The fastest oligonucleotide based artificial nuclease up to date is a peptide nucleic acid © XXXX American Chemical Society

Received: October 2, 2015 Revised: November 4, 2015

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DOI: 10.1021/acs.bioconjchem.5b00534 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

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RESULTS AND DISCUSSION One of our methods used to investigate and quantify the RNA cleavage process is based on anion exchange HPLC (IEHPLC). This technique is fast and proven to be accurate.29,30 In combination with mass spectrometry (ESI-TOF) it characterizes the RNA fragments which are products of the cleavage and defines precisely the positions of phosphodiester bond breakage. Another method which finds application in RNA cleavage studies is based on polyacrylamide gel electrophoresis. Cy5-labeled oligonucleotide fragments are separated by denaturating PAGE on a DNA sequencer and comparison with the hydrolysis ladder of the substrate defines the position where cleavage occurs.31 Both techniques imply integration of peak areas to quantify the degraded RNA. To confirm the consistency of the methods, cleavage experiments with electrophoretic analysis were performed using PNAzyme 1 (Chart 1) which is the fastest artificial

Figure 1B. Here all fragments are separated and analyzed with MS in order to identify the cleavage position. The cleavage rates obtained with RNA substrates 3 and 7 are in the same range (somewhat lower for RNA 3, but the substrates are also not identical) and the single cleavage sites are identical. The data shown in Figure 1 correspond to buffers prepared from Milli-Q water. A standard procedure to inactivate traces of natural nucleases is to treat such water with diethyl dicarbonate and to autoclave it. This procedure had no influence on the rates of PNAzyme 2. However, it severely retards the reaction of PNAzyme 1. Thus, even minor changes in the experiment may have considerable impact on the absolute values of reaction rates (for a discussion see Supporting Information). The strength of the electrophoretic analysis is the straightforward assignment of the cleavage positions as the electropherogram shows only the Cy5-labeled RNA fragments while the benefit using IE-HPLC in combination with ESI-TOF is the unequivocal identification of all cleavage products and the ability to distinguish between 2′,3′-cyclic phosphates and the products of further hydrolysis (2′- and 3′-phosphates). After this comparison we then used both methods for the analysis of RNA hydrolysis by the newly designed PNA based metal-free cleaver, PNAzyme 2. To test the tris(2-aminobenzimidazole) conjugate 2, six oligonucleotide RNA substrates 3, 4, and 7−10, that should form tetranucleotide bulges upon hybridization, are used (Chart 1). The first studies with the Cy5-labeled RNA target 3 were conducted with equimolar amounts of conjugated PNA and substrate at pH 7.4 (Figure 2A). What can be seen in the initial experiments is that RNA cleavage is confined to the bulge region and occurs mainly after nucleotides U9, A10, and A11. The data obtained with the sequencer is in agreement with the result from IE-HPLC (Figure 2B). The chromatogram shows 6 major fragment peaks, 3 of which come early and correspond to the shorter products of cleavage and 3 are eluted later and relate to the longer fragments. Both methods reveal that PNAzyme 2 does not have a preferred cleavage position, but tend to cause bond breakage throughout the entire bulge region of the RNA substrates 3 and 7. Cleavage rates are also comparable for the two substrates (t1/2: 23 and 17 h respectively; Table 1, entries 9 and 7). After prolonged incubation time, a secondary effect can be observedcleavage at position C7, probably due to subsequent cleavage of fragments which are still bound to the PNA (Figure S1). To exclude that the results are caused by contamination with natural nucleases, studies were also performed with noncomplementary RNA sequences 5 and 6 which were not cleaved at all (Figure S2). The experiments show that hybridization of PNA and RNA is an absolute requirement for substrate cleavage. To examine the effect of the pH on PNAzyme 2 efficacy, cleavage of RNA substrate 3 was carried out also at pH 8. The cleavage pattern is unchanged, and as found in earlier studies with tris(2-aminobenzimidazole) based systems, cleavage gets faster with increasing pH (Table 1, entry 10). Rates seen in analogous experiments with RNA 7 and IE-HPLC analysis are somewhat higher (entries 2−7). However, they consistently show increasing rates with raising pH. The buffer type has no major influence. Thus, imidazole does not act as a cocatalyst. Kinetic runs with substrates 3 and 4, however, tend to be slightly faster in Tris than in HEPES buffer. Addition of Cu2+ ions (10 μM) substantially retards the RNA cleavage reaction

Chart 1. Sequences of PNAzymes 1 and 2 and Oligonucleotides 3−10a

a

Underlined areas are noncomplementary regions where bulge formation is expected.

nuclease system currently reported.21 The assay is performed at conditions as close as possible to the originally reported conditions. The DNA sequencer assay requires Cy5-labeling of the RNA for detection and the presence of T10- and T4-DNA spacers to obtain full separation of all possible fragments from the substrate strand. Thus, the substrates for this analytical technique are sequences 3−6 (Chart 1). Due to the modifications applied, the analysis of RNA target 3 shows a clear electropherogram, where the single cleaved phosphodiester linkage can be readily identified by direct overlay and comparison with the hydrolysis ladder of the substrate. As visible in Figure 1A, the cleavage occurs in the bulge region between nucleotides A10 and A11. The results correlate well with the data obtained from IE-HPLC shown in B


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Figure 1. Comparison of two techniques showing site-specific cleavage of the target RNAs 3 and 7 induced by PNAzyme 1. A: 4 μM of RNA target 3, 4 μM PNAzyme 1, 10 mM HEPES buffer, pH 7.4, 0.1 M NaCl, 37 °C, 20 μM Cu2+, (a) 90 min incubation of 1 and 3, (b) hydrolysis ladder of substrate 3. B: 4 μM of RNA target 7, 4 μM PNAzyme 1, 10 mM HEPES buffer, pH 7.4, 0.1 M NaCl, 37 °C, 10 μM Cu2+, 80 min (G•T − indicates a wobble base pair).

Figure 2. Comparison of two techniques examining the cleavage of the target RNAs 3 and 7 induced by PNAzyme 2. A: 4 μM of RNA target 3, 4 μM PNAzyme 2, 50 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 37 °C, (a) 0 min, (b) 24 h, (c) 40 h (75% cleaved RNA target 3), (d) hydrolysis ladder of substrate 3. B: 4 μM of RNA target 7, 4 μM PNAzyme 2, 10 mM HEPES buffer, pH 7.4, 100 mM NaCl, 37 °C, 24 h (G•T − indicates a wobble base pair).

(Table 1, entry 1) suggesting that it may interfere with the tris(2-aminobenzimidazole) catalyst. PNAzyme 1 works best with RNA substrates characterized by an AUAA bulge followed by a GT wobble base pair.21 To see if PNAzyme 2 has identical preferences, we changed the bulge sequence from AUAA to AAAA (RNA substrate 4; Table 1, entry 12; Figure S5). In contrast to the Cu2+-phenanthroline system, faster cleavage was observed, bringing down the difference from the metal ion system to only 1 order of magnitude and to a rate comparable to those of reported Zn2+phenanthroline PNAzymes.20 The influence of bulge sequences on the cleavage rates was further investigated with three additional RNA sequences 8−10. These were subjected to PNAzyme 2 at pH 7 and revealed, as already found with substrate 4, that the A4 bulge (RNA substrate 8) was more susceptible to cleavage (Table 1, entry 11; Figure 3). A substrate with two uridines in the bulge (RNA substrate 9, entry 13) was cleaved at a rate similar to that of RNA 7. If the GT wobble pair closing the bulge was exchanged by AT (RNA substrate 10), the rate dropped further. The higher rate with the A4 bulge is possibly due to the better stacking of the four adenines with the cleaving catalyst.

Table 1. Cleavage Rates and Substrate Half Lives Observed with PNAzyme 2 entry

RNA

experimental conditions

k (min−1)

t1/2 (h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

b

10 μM Cu , HEPES buffer pH 7 1 M imidazole buffer pH 6.4 1 M imidazole buffer pH 7 1 M imidazole buffer pH 7.4 HEPES buffer pH 6.5 HEPES buffer pH 7 HEPES buffer pH 7.4 HEPES buffer pH 7.4 Tris buffer pH 7.4 Tris buffer pH 8 HEPES buffer pH 7 Tris buffer pH 8 HEPES buffer pH 7 HEPES buffer pH 7

0.00034 0.00040 0.00058 0.00068 0.00041 0.00066 0.00067 0.00042 0.00051 0.00074 0.0013 0.00097 0.00067 0.00046

34 29 20 17 28 17 17 28 23 16 9.1 12 17 25

7 7b 7b 7b 7b 7b 7b 3a 3a 3a 8b 4a 9b 10b

2+

a

Analysis by gel electrophoresis. bAnalysis by IE-HPLC. For details see Figure 2. Buffer concentration: HEPES 10 mM, Tris 50 mM. All experiments were run in duplicate or triplicate.

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identification, while the electrophoretic method has its strengths to visualize reactions leading to multiple fragments. It is also sensitive to small changes in the cleavage pattern that are hardly visible in the HPLC/MS approach. There are differences in kinetics, perhaps mainly due to the different substrates needed, which may require slightly different conditions. In spite of this experimental discrepancy, reaction rates nevertheless are in a similar range and there is a clear consistency between the methods. The newly designed tris(2-aminobenzimidazole) based PNAzyme 2 is demonstrated to be an artificial nuclease with turnover of RNA substrate. This nonmetal ion artificial nuclease preferably cleaves in the bulge region formed upon hybridization to the RNA substrate and displays sensitivity to both bulge sequence and to the bulge closing base pair. However, it does not show the pronounced sequence and site preference of the Cu2+-phenanthroline based PNAzyme 1 (Figure 1). Although there is still some way to go with metal ion free nucleases like PNAzyme 2, these systems are approaching the rates of metal-ion based artificial nucleases and may already be considered for some biotechnological applications.

Figure 3. Extent (%) of uncleaved RNA substrates 8−10 as a function of time (as analyzed by IE-HPLC). Conditions: 4 μM of RNA targets 8, 9, and 10, 4 μM PNAzyme 2, 10 mM HEPES buffer, pH 7, 100 mM NaCl, 37 °C. Half-lives for substrates 8, 9, and 10 are about 9, 17, and 25 h, respectively.

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The PNAzyme 2 was then tested for catalytic turnover activity in order to establish if it can be considered a true catalyst, i.e., artificial nuclease. PNAzyme 2 was incubated with a 5-fold excess of RNA substrate 3 for a period of 2 weeks. The result shows that after the first week most of the substrate was degraded (Figure 4 and Figure S3 in SI). The curve of substrate

MATERIALS AND METHODS Peptide nucleic acid monomers were purchased from Link technologies and Rink Amide resin from Biotage. HBTU and HOBt were purchased from Novabiochem. Solvents and reagents for solid-phase synthesis were synthesis grade from Applied Biosystems and IRIS Biotech Gmbh (Germany). Other solvents were purchased from Merck Eurolab. High-resolution mass spectrometry (HRMS) was performed on a Micromass LCT electrospray time-of-flight (ES-TOF) mass spectrometer in acetonitrile−water 1:1 (v/v) solutions (Huddinge group). The molecular weights of the oligoribonucleotide and peptide nucleic acid conjugates were reconstructed from the m/z values using the mass deconvolution program of the instrument (Mass Lynx software package). MALDI: Applied Biosystems/ Perseptive Biosystems Voyager-DE STR (Frankfurt group). The RNA substrates 7−10 were purchased from Thermo Scientific. They were first purified by semipreparative IE-HPLC (ion exchange high performance liquid chromatography) and then with RP-HPLC. Concentrations of both RNA and PNA were determined by UV absorption at 260 nm and calculated from extinction coefficients obtained by the nearest-neighbor approximation. Chemicals for polyacrylamide gel electrophoresis were purchased from Roth (Karlsruhe, Germany). Oligonucleotides 3−6 were obtained from Biospring (Frankfurt, Germany). Cy5 NHS ester was purchased by Lumiprobe (Hannover, Germany). RNA substrates 3 and 4 were labeled by us. All chemicals used in the kinetics experiments were of molecular biology grade. Synthesis of PNAs. The PNA sequence was assembled automatically on a solid support (Rink Amide resin) using the manufacturer’s protocol for the Alstra Initiator Biotage peptide synthesizer with 9-fluorenylmethyloxycarbonyl (Fmoc)-chemistry and N,N′-diisopropylcarbodiimide (DIC) as coupling agent and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) as additive. PNA building blocks were from Link Technologies Ltd. (Strathclyde, UK), Fmoc-αN-Lys(εN-Boc)OH and FmocMtt-L-(S)-diaminopropionic acid from Iris Biotech Gmbh (Marktredwitz, Germany). Synthesis of PNAzyme 2. The Nβ-methyltrityl protection on the diaminopropionic acid was removed by subjecting 20 mg of the solid supported PNA to 1% trifluoroacetic acid in

Figure 4. Turnover kinetics of substrate 3 in the presence of conjugate 2. Conditions: 4 μM substrate 3, 0.8 μM conjugate 2, 50 mM TrisHCl, pH 8, 100 mM NaCl, 37 °C.

concentration versus time shows a straight line as long as substrate is present in excess (saturation of the catalyst) and develops the characteristics of first order kinetics only in the last phase of the reaction. Thus, PNAzyme 2 is a nonmetal ion based artificial nuclease for sequence selective cleavage of RNA.

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CONCLUSIONS In the present study we have compared two analytical methods to determine the cleavage kinetics of RNA catalyzed by PNA based artificial nucleases. The two methods give comparable results with respect to cleavage sites. The IE-HPLC/MS approach has the advantage of unequivocal fragment D


Article

Bioconjugate Chemistry DCM for 5 × 1 min, followed by washing with DCM and NMP. Then, the trisaminobenzimidazole carboxylic acid (11.3 mg, 21 μmol, 5 equiv) cleaver27,28 was dissolved in 105 μL NMP and HOBt (2.84 mg, 21 μmol), NEt3 (12 μL, 84 μmol) and DIC (3.3 μL, 21 μmol) were added. The solution was shaken for 5 min and then added to the resin bound PNA. The mixture was shaken at RT for 72 h. The resin was washed with NMP, DCM, and MeOH 5 times, respectively, dried in vacuum and treated with 1.5 mL of TFA/TIS/water (95/2.5/2.5) (200 μL) mixture for 2 h. The product was purified by HPLC on a Phenomenex Gemini C18 column (150 × 4.6, 5 μm), linear gradient of 0−50% MeCN in 0.1% TFA for 40 min, 1 mL/min, 50 °C, 254 nm, tR = 16.3 min. MS (MALDI) m/z = 3810.2 [M + H]+, calcd for C158H197N79O38+H+: 3809.6. RNA Cleavage Assay Using IE-HPLC. The reactions were performed in 0.3 mL glass tubes with screw cap immersed in a water bath at 37 °C. Incubation was carried out in a buffer with the following recipe: 10 mM Hepes, 100 mM NaCl at the pH value described. All reactions were performed in equimolar concentrations of substrate RNA and PNAzyme. The samples were prepared at room temperature by adding the buffer, RNA and the amount of water necessary to achieve the right concentration, incubating the mixture for 10 min and then adding the aliquot from the PNAzyme stock solution. The reaction mixture was then placed in the water bath. The final volume of the sample was 160 μL and 4 aliquots were collected at specific times (40 μL each one) and quenched with 70 μL of 300 μM EDTA solution. The samples were frozen and stored at −18 °C until being analyzed by HPLC. Inactivation of RNases. The following experimental steps (for purification of Cy5-labeled oligonucleotides and conjugates, polyacrylamide gel electrophoresis and the RNA cleavage assay using the sequencer) were performed under sterile conditions. Plasticware, tubes, and most solutions were treated with diethylpyrocarbonate (DEPC). Solutions which are not compatible with DEPC treatment were prepared by mixing up molecular biology grade powdered reagents in DEPC-treated ultrapure water (Note: Experiments with PNAzyme 1 require solutions untreated with DEPC!). All glassware was baked at 180 °C for 6 h. Purification of Cy5-Labeled Oligonucleotides and Conjugates. The oligonucleotides were purified by denaturing PAGE (16% monomer, 7 M urea). The bands of interest were excised, the gel fragments transferred to a nuclease-free tube and submerged with elution buffer (500 mM NaOAc, 0.1% SDS, 2 mM EDTA). We routinely incubated the gel fragments under vigorous shaking overnight at room temperature. Quantum Prep Freeze’N′Squeeze spin columns (BioRad, Munich, Germany) were used to remove the gel fragments. After EtOH precipitation, the pellet was dissolved in 1 mL DEPC-treated H2O and desalted on a NAP-10 column. The pooled fractions were lyophilized to dryness (dried on a speedvac), and the pellet was dissolved in DEPC-treated H2O to give a concn of approximately 0.8 μg/μL. RNA Cleavage Assay Using Gel Electrophoresis. Four μM Cy5-labeled RNA was incubated with the indicated conjugate concentration (0.8−4 μM) in a 50 mM Tris buffer (or 10 mM HEPES if stated otherwise) at pH 7.4 and pH 8 (if not indicated otherwise) containing 100 mM NaCl. All cleavage reactions were performed at 37 °C, time as indicated. Prior to gel electrophoresis, the samples were diluted with DEPCtreated water to 150 nM, which is optimal for analysis on the sequencer.

Polyacrylamide Gel Electrophoresis. The oligonucleotide fragments were separated by denaturing PAGE (16% monomer, 7 M urea) on a DNA sequencing device (ALF express, GE Healthcare/Amersham Biosciences). Prior to electrophoresis, each sample was completed with 1.5 volumes of loading buffer (8 M Urea, 20 mM EDTA and 0.2% Crocein Orange in DEPC water) and 5 μL were loaded on the gel. The following running conditions were chosen: 1500 V (maximum), 60 mA (maximum), 25 W (constant), 57 °C, 2 s sampling interval, and 300 min running time. For analyzing the electropherograms the AlleleLinks 1.01 software package (Amersham Biosciences, Uppsala, Sweden) was used. The peak areas under the curves were added up, and the percentage of degraded RNA was calculated. Multiple cleavage reactions were disregarded in this system. All data were averaged over a minimum of two experiments.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00534. Electropherograms; graphs displaying cleavage kinetics; a discussion of DEPC effects; characterization of PNAzyme 2 by HPLC and mass spectrometry (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

Plamena Dogandzhiyski and Alice Ghidini contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding from the Swedish Research Council and the European seventh framework program (238679, PhosChemRec) is gratefully acknowledged.

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REFERENCES

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Studies on Tris (2-aminobenzimidazole)-PNA Based Artificial

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