ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 6, pp. 626–630. © Pleiades Publishing, Ltd., 2015. Original Russian Text © V.A. Spiridonova, A.M. Kudzhaev, A.V. Melnichuk, A.A. Gainutdinov, A.G. Andrianova, T.V. Rotanova, 2015, published in Bioorganicheskaya Khimiya, 2015, Vol. 41, No. 6, pp. 696–700.
Interaction of DNA Aptamers with the ATPDependent Lon Protease from Escherichia coli1 V. A. Spiridonovaa, A. M. Kudzhaevb, A. V. Melnichuka, A. A. Gainutdinova, A. G. Andrianovab, and T. V. Rotanovab, 2 aMoscow
State University, Moscow, 119899 Russia Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117871 Russia
b
Received May 26, 2015, in final form, June 8, 2015
Abstract—ATPdependent Lon protease of E. coli (EcLon) is a key enzyme of the quality control system of the cell proteome. The EcLon subunit comprises Nterminal noncatalytic region, ATPase module and pro teolytic domain (serine–lysine endopeptidase). A distinctive feature of the EcLon is its ability to interact with DNA, however either DNA binding site(s) or the role of the complex EcLon/DNA have not yet been characterized. Aptamers, small nucleic acids with high specificity to organic compounds of different natures, are known to be a promising tool for the study of molecular mechanisms of interaction between nucleic acids and protein ligands. EcLon protease was found to form complexes with the previously obtained thrombin aptamers whose molecules comprise the duplex domains and Gquadruplex region. The aptamer affinities to the enzyme have been characterized. The synthesis of novel aptamers specific to EcLon protease is planned for studying the mechanism of the enzymeDNA complexation. Keywords: ATPdependent Lon protease, DNA binding, DNA aptamers, E. coli DOI: 10.1134/S1068162015060151
FORMULATION OF THE PROBLEM ATPdependent LonA protease from E. coli (Ec Lon) and its homologues in bacteria and eukaryotes are the key enzymes in the quality control system of the cell proteome, including molecular chaperones and energydependent peptidehydrolases [1–3]. The components of the quality control system ensure the stability of the cell proteome in all natural kingdoms via correct folding of proteins, prevention of the for mation of protein aggregates and selective degradation of regulatory proteins and also abnormal, damaged or mutated proteins. EcLon is a bifunctional homohexameric enzyme, its subunit comprises an Nterminal noncatalytic region, twodomain ATPase module, and a proteolytic domain (serine–lysine endopeptidase) [4]. A distinc tive feature of the EcLon protease is its ability to interact with DNA. Whereas, either binding site(s) of nucleic acid (NA) or the role of the ЕсLon·DNA complex have not yet been characterized [5–8]. Abbreviations: Apt, aptamer; NA is nucleic acid; CCM, chemi cal cleavage of mismatch; SELEX, systematic evolution of ligands by exponential enrichment. 1 The article has been adapted from the report presented at the VIIth AllRussian symposium “Proteins and Peptides”, Novosi birsk, 12–17 July, 2015. 2 Corresponding author: phone: +7 (495) 3354222; fax: +7 (495) 3357103; email:
[email protected].
Aptamers, i.e., small nucleic acids with high speci ficity to organic compounds of different natures, have been recently used as a promising tool for the study of molecular mechanisms of interaction between nucleic acids and ligands, including protein ligands. Aptamers are singlestranded DNA/RNAbased molecules with a length of 40–100 nucleotides with rather complex threedimensional structure [9]. This structure of aptamers confers them the binding specificity to dif ferent ligands. The SELEX (systematic evolution of ligands by exponential enrichment) method devel oped in 1990 independently in two laboratories is used for the selection of highly specific aptamers [10, 11]. The SELEX process starts from generating a ran domized DNA sequence library, which is composed of 30–60 nucleotide random sequence at the center flanked by necessary primer binding sites at the 5' and 3' ends. The randomized DNA sequence library is generated through synthesizing an oligonucleotide sequences pool with an automated DNA synthesizer by attachment of four phosphoramidite activated monomeric units (synthons) at each coupling stage. Even so the library can generate Nmax= 4n different sequences from n nucleotides, functional target mole cules comprise only a small portion: 1 aptamer is pro duced in 109–1013 molecules. The target aptamers which can bind to the target molecule are then selected through separation of
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(а)
(b) 5'
5'
A2 С3
T2 С29 T28
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G10 G6
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Fig. 1. (a) The nucleotide sequence (5'–3') and (b) the secondary structure of DNA aptamers [13, 14] used in the study of their interaction with the LonS679А protease.
aptamertarget complexes from unbound nucleic acids by affinity chromatography, gel electrophoresis, or other methods. After several rounds of selection aptamerenriched fraction is generated that binds to the target by several orders of magnitude more effec tive than oligonucleotides of the original library. The affinity of aptamers obtained by SELEX to target pro teins is typically comparable to the affinity of natural antigenantibody complexes [9, 12]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
The aim of this work was to study the possibility of a fullsized multidomain EcLon protease interac tion with small singlestranded DNA. DNAbased aptamers were selected using a convenient model based on the selective affinity for thrombin [13, 14]. MATERIALS AND METHODS The reagents were purchased from Sigma, Aldrich, BioRad (United States), Fluka (Switzerland), Boe Vol. 41
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1 2 3
(b) 4 5 6 7 8
Complex
Aptamer
(c) 1 2 3 4 5 6 7 8 9 10 Complex
Complex
Aptamer
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Fig. 2. The formation of LonS679A complexes with (a) 31ТВА, (b) RE31 and (c) ST43 aptamers (gel electrophoresis in 8% PAAG). Lanes: 1, nucleotide markers, 2, aptamer (control), 3–10, samples prepared by incubating the LonS679A and aptamer (4°C, 1 h) in different ratios (see concentrations in the table).
hringer Mannheim (Germany), Pharmacia (Sweden), Difco (England), Panreac (Spain), Fermentas (Lithuania), and Reakhim (Russia). Mutant form of EcLon protease, LonS679A was prepared as previously described in [21]. The final stages of affinity chromatography on Nisepharose and gelfiltration were performed on columns pur chased from GE Healthcare: HiTrapTMFF (2 × 5 mL tandem) and Sephacryl S400 (volume of 120 mL), respectively. Standard analytical methods. Protein concentra tion was determined by Bradford’s method [22]. The homogeneity of the protein in preparations was checked electrophoretically [23] using a commercial kit of markers (M, kDa) that contains βgalactosidase (116.0), bovine serum albumin (66.2), ovalbumin (45.0), lactate dehydrogenase (35.0), restrictase Bsp98I (25.0), βlactalbumin (18.4), and lysozyme (14.4). Oligonucleotidesaptamers (Fig. 1a) were synthe sized according to the phosphoramidite method (Syn thol, Russia). LonS679A·aptamer complexes preparation. Sam ples of nucleotides were heated for 2 min at 100°С, rapidly cooled in an ice bath and incubated with Lon S679A in 50 mM TrisHCl buffer at pH 7.5 containing 140 mM NaCl and 5 mM KCl for 60 min at 4°С (aptamers and protein concentrations are given in the legend to Fig. 2). Aliquots of the reaction mixtures were subjected to nondenaturing gelelectrophoresis. SYBR GREEN 1 fluorescent dye was used to visualize zones containing DNA aptamer (in case of 15ТВА, radiolabeled aptamer prepared following the known procedure was used [24]). RESULTS AND DISCUSSION Along with the biologically active Bform of DNA with common features of its structure independent of
the nucleotide sequence, DNA can assume a wide variety of noncanonical structures. Their stability and structural features depend on the types and the sequence of the heterocyclic bases. The most interest ing forms of DNA are Grich structures referred to as Gquadruplexes [15], which are the subject of inten sive research and attract increasing interest, mainly, in connection with their supposed participation in the most important biological processes and the associ ated human disease [16, 17]. In the present study we have investigated ability of the EcLon protease to interact with nucleic acid frag ments at the example of proteolitically inactive mutant of the enzyme by catalytic residue Ser679 (Lon S679A) [18] using a series of thrombinspecific DNA aptamers: 15ТВА, 31ТВА, RE31, ST43, and RE15T which were characterized in [13, 14] (Fig. 1a). In this group, the nucleotide sequence of the aptamer 15ТВА generates true Gquadruplex, which is formed by two Gtetrads connected by small loops (Fig. 1b). Oligo nucleotides 31ТВА, RE31 and ST43 are twodomain structures containing common with 15ТВА Gqua druplex domain and different duplex domains. RE15T is a duplex analogue of RE31, wherein the central 15 unit Gquadruplex is substituted by a nucleotide sequence of 15 thymidine residues (Fig. 1a). The formation of nucleoprotein complexes of Lon S679A with aptamers was visualized electrophoreti cally using either radiolabeling of DNA (in the case of the 15ТВА aptamer) or staining with dye SYBR GREEN 1 (for other aptamers) [19]. It was found that the Lon protease does not show a strong ability to bind to any individual Gquadruplex (15ТВА) or duplex aptamer (RE15T). However, Lon S679A forms complexes with twodomain 31ТВА, RE31 and ST43 aptamers (Figs. 2a–2c). Fig. 2 shows that in each case the incubation of aptamer with increasing amounts of the target protein leads to enhancement of fluorescence intensity of the com
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Efficiency of aptamer interaction with LonS679A protease (Aptbound is aptamer bound to the enzyme) Aptamer [Apt], nM 31 TBA, 87.5
RE31, 180.0
ST43, 125.0
Number of lane in Fig. 2
LonS679A, nM
Proportion of Aptbound
2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 2 3 4 5 6 7 8 9 10
– 43.75 87.50 131.25 175.00 218.75 262.50 350.00 437.50 – 140.50 281.00 421.50 562.00 702.50 843.00 – 62.50 125.00 187.50 250.00 312.50 375.00 500.00 625.00
– – 0.156 0.293 0.365 0.339 0.472 0.520 0.533 – – – 0.368 0.376 0.445 0.476 – – – 0.255 0.359 0.440 0.541 0.625 0.635
Relative binding efficiency, %
98.5
68.0
100.0
See the materials and methods section for conditions of the complexes preparation.
plex’s band on the electrophoregram. It should be noted that the time of the experiment (1 h) was insuf ficient to reach equilibrium in the complexation pro cess. This did not allow calculating the dissociation constant of the LonS679A·aptamer complexes. A measure of the efficiency of nucleic acid interac tion with the enzyme in each experiment can be the proportion of aptamer bound to Lon protease (table) determined as the ratio of the Lon protease–aptamer complex zone fluorescence intensity to the total fluo rescence of the complex and free aptamer in the cor responding lane of electrophoregram (Fig. 2). Com parison of these characteristics for various aptamers that form complexes with the enzyme at similar con centrations (for example, about 400 nM), makes it possible to compare affinity of nucleic acids to the Lon protease. It follows from data in the table (the lines in bold) that 31ТВА and ST43 aptamers exhibit close affinity to the enzyme, while the ability of RE31 to bind with Lon protease is significantly reduced. Hence, this study has shown that the ATPdepen dent Lon protease from E. coli is able to form com plexes with a number of known twodomain DNA RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
aptamers. This research is supposed to be continued via selection of aptamers with high specificity to the Lon protease, which will open prospects for the use of new aptamers in the study of the interaction mecha nism of the enzyme with nucleic acids and in the study of the structure of its DNA binding site(s). ACKNOWLEDGMENTS The authors would like to thank Doctor of Chem istry N.L. Eremeev (Moscow State University, Mos cow) for the helpful discussion of the results. This work was supported by the Federal Target Program “Research and Development on Priority Directions of ScientificTechnological Complex of Russia for 2014–2020”, No. 14.616.21.0011. REFERENCES 1. Buchberger, A., Bukau, B., and Sommer, T., Mol. Cell, 2010, vol. 40, pp. 238–252. 2. Mogk, A., Haslberger, T., Tessarz, P., and Bukau, B., Biochem. Soc. Trans., 2008, vol. 36, pp. 120–125. Vol. 41
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Translated by M. Novikova
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