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Rnase T1 Mimicking Artificial Ribonuclease N 2356–2367 Nucleic Acids Research, 2007, Vol. 35, No. 7 Published online 27 March 2007 doi:10.1093/nar/gkm143 RNase T1 mimicking artificial ribonuclease N. L. Mironova, D. V. Pyshnyi, D. V. Shtadler, A. A. Fedorova, V. V. Vlassov and M. A. Zenkova* Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave., 8, Novosibirsk, Russia, 630090 Received December 21, 2006; Revised February 5, 2007; Accepted February 22, 2007 ABSTRACT transesterification reaction [for example, imidazole (9–11), aminogroups (12), guanidinium groups (13)]. aRNases Recently, artificial ribonucleases (aRNases)—con- designed using this approach usually display cleavage jugates of oligodeoxyribonucleotides and peptide specificity similar to that of RNase A: they cleave RNA (LR)4-G-amide—were designed and assessed in predominantly at linkages within Pyr-A motifs, which are terms of the activity and specificity of RNA clea- known to be highly sensitive towards various cleaving vage. The conjugates were shown to cleave RNA at agents (14). These aRNases accelerate cleavage at the Downloaded from Pyr-A and G–X sequences. Variations of oligonu- most sensitive sites within RNA. cleotide length and sequence, peptide and linker The sequence specificity of natural RNases is deter- structure led to the development of conjugates mined by the substrate recognition centre in which the exhibiting G–X cleavage specificity only. The most specific interaction of amino acids with RNA provides efficient catalyst is built of nonadeoxyribonucleo- for the specific binding and placement of a particular http://nar.oxfordjournals.org/ heterocyclic base, thus resulting in the optimal conforma- tide of unique sequence and peptide (LR)4-G-NH2 connected by the linker of three abasic deoxyribo- tion of internucleotide phosphodiester bonds, which are subjected to cleavage. Accurate mimicking of ribonuclease nucleotides (conjugate pep-9). Investigation of the active centres is a difficult task because of their complex cleavage specificity of conjugate pep-9 showed spatial structure providing for multipoint contacts within that the compound is the first single-stranded the enzyme–substrate complex, and specific and dynamic guanine-specific aRNase, which mimics RNase T1. nature of the centres that undergo conformational Rate enhancement of RNA cleavage at G–X linkages changes. catalysed by pep-9 is 108 compared to non- Attempts were made to stabilize the RNA heterocyclic by guest on April 16, 2015 catalysed reaction, pep-9 cleaves these linkages bases optimally for cleavage conformations via stacking only 105-fold less efficiently than RNase T1 interactions with aromatic amino acids [for example, 5 (kcat_RNase T1/kcat_pep-9 ¼ 10 ). phenylalanine (15)], which were introduced into the structure of conjugates mimicking RNase active centres. However, these conjugates did not exhibit any INTRODUCTION cleavage specificity other than Pyr-A. aRNases displaying other specificity were developed by introducing guanidi- The major challenges in the development of artificial nium groups and arginine residues into the structure ribonucleases (aRNases) are the achievement of sequence of aRNases: some G–X cleavage activity was reported specificity of RNA cleavage and high cleavage efficiency. for conjugates of anthraquinone and imidodiacetate Natural ribonucleases of RNase A family (1–4) exhibit bearing carboxylic and ammonium ions (13) and pyrimidine-X specificity. Ribonucleases of T1 family conjugates of oligodeoxyribonucleotides and peptide (RNase T1 from Aspergillus oryzae (5) and RNase F1 (LR)4-G-amide (16–21). from Fusarium moniliforme (6) exhibit guanine-X Recently, the conjugates of peptide (LR)4-G-NH2 specificity, and RNase U2 from Ustilago sphaerogena attached to 50-terminal phosphate of antisense oligonu- (7) exhibit adenine-X specificity. Attempts have been cleotides were obtained (16,19). These conjugates were made to alter the specificity of ribonuclease T1 by protein found to cleave RNA both in the vicinity of oligonucleo- engineering methods (8), but this goal has not been tide complementary sequence and in a random manner achieved till date. at Pyr-A and G–X linkages (16,19). We found that the One approach to the design of aRNases consists of oligonucleotide in the conjugates plays an unusual role: it mimicking active sites of natural enzymes by conjugates promotes formation of an ‘active’ peptide conformation bearing the functional groups of amino acids that form because the peptide itself exhibits no ribonuclease activity the catalytic centre of the enzyme and catalysing the (17,20). All designed oligonucleotide–peptide conjugates *To whom correspondence should be addressed. Tel: (383)3333761; Fax: (383)3333677; Email: [email protected] ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2007, Vol. 35, No. 7 2357 50 30 displayed either G–X4Pyr-A or G–X5Pyr-A activity, (pdRib)3- pTCTCTT (pep-TCTCTT), NH2-G-(RL)4- 50 30 (þ50G) but both activities were observed simultaneously. The (pdRib)3- pGGGATCTCTT (pep-9 ), NH2-G- 50 30 (À50G) main task of this work was to design the conjugate(s) (RL)4-(pdRib)3- pGATCTCTT (pep-9 ), 50 30 exhibiting G–X cleavage activity similar only to NH2-G-(RL)4-(pdRib)3- pGGA-(pdRib)4-T (pep- 50 30 RNase T1. GGA(pdRib)4T) and NH2-G-(RL)4-(pdRib)3- pGGAT In this article, we solve this problem and describe the (pep-GGAT) were synthesized by the formation of first single-stranded guanine-specific aRNase—conjugate phosphamide bond between 50-terminal phosphate of of nonadeoxyribonucleotide GGATCTCTT and peptide deoxyribose linker or 50-phosphate group and N-terminal (RL)4-G-amide connected by the linker of three deoxy- a-amino group of oligopeptide according to published ribose residues (pep-9), which display only G–X cleavage protocol (22). The conjugates were isolated by reverse- activity under various conditions. Rate enhancement of phase HPLC on LiChrosorb RP-18 column (4 Â 250 mm). RNA cleavage at G–X linkages catalysed by pep-9 is 108, Homogeneity of the conjugates was tested by electro- as compared to non-catalysed reactions; pep-9 cleaves G– phoresis in 15% polyacrylamide/8 M urea gel followed X linkages only 105-fold less rapidly than RNase T1 by staining with ‘Stains-all’. The homogeneity of the 5 (kcat_RNase T1/kcat_pep-9 ¼ 10 ). conjugates was 95–98%. The structure and properties of the conjugate pep-9 are protected by patent (23). MATERIALS AND METHODS MALDI-TOF mass spectrometry Downloaded from [g-32P]ATP (specific activity 43000 Ci/mM) was from Molecular weights of peptides, oligonucleotides and Biosan Co. T4 polynucleotide kinase and RNase T1 conjugate pep-9 have been verified by MALDI-TOF were purchased from Fermentas (Lithuania). Peptide mass spectrometry on a REFLEX III mass spectrometer (LR)4-G-NH2 was purchased from Diapharm Ltd. equipped with a pulsed nitrogen laser emitting at 337.1 nm (St. Petersburg, Russia). Fok I restriction endonuclease (Bruker Daltonics, Germany). An overlayer preparation was from Sibenzyme (Russia). T7 RNA-polymerase was used with a 3-hydroxypicolinic acid (HPA) or http://nar.oxfordjournals.org/ was prepared by Dr V. Ankilova (this institute). All 2,5-dihydroxybenzoic acid (DHB) as matrix. A 10:1 buffers were prepared using MilliQ water, contained mixture 50 mg/ml matrix in aqueous acetonitrile 0.1 mM EDTA and were filtered through 0.22-mm (1:1 v/v), and 100 mg/ml aqueous ammonium citrate was millipore filters. spotted in 1 mL aliquots on a bed and dried. Here, 1 ml 10 mM aqueous solution of a sample was added. MALDI- Oligonucleotides TOF mass spectra were acquired in positive- or negative- 0 ion mode. Samples were analysed in reflector mode using Oligoribonucleotides 5 AGAAACAACGGUUCGGAAG 0 0 an accelerating voltage of 20 kV. All spectra were the UU3 (RNA-21s) and 5 CUAACUUCCGAACCGUU by guest on April 16, 2015 0 result of signal averaging 200 laser shots. Calibrations GUUU3 (RNA-21as), the chimeric ribo/20-O-methylribo 50 30 were performed in accordance with the manufacturer’s oligonucleotides UUC AUrGUAAA (RNA-10rGU), 50 30 50 recommendations. For oligonucleotides, peptide and UUCAUrGAAAA (RNA-10rGA), UUCAUrG 30 50 30 conjugate pep-9 expected and measured masses were: GAAA (RNA-10rGG) and UUCAUrGCAAA (pdRib)3-GGATCTCTT expected 3326.1, measured (RNA-10rGC) were chemically synthesized by standard 3326.73, a [MþH]þ; peptide (LR) -G-NH expected protocols. 20-O-Methylribo units are underlined. 4 2 50 30 1151.78, measured 1151.81, m [MþH]þ; conjugate pep-9 Oligodeoxyribonucleotides (pdRib)3- pGGATCTCTT , 50 3 50 30 expected 4459.6, measured 4459.03, a [MþH]þ; conjugate pGGATCTCTT , (pdRib)3- pGGATCTCTA , (ÀL) 50 30 50 30 50 pep-9 expected 3919.2, measured 3922.55, a [MþH]þ; (pdRib)3- pTCTCTC , pTCTCTC , (pdRib)3- pTCT 30 50 30 50 where a, m refers to average and monoisotopic mass, CTT , (pdRib)3- pGGGATCTCTT , (pdRib)3- pGAT 30 50 30 respectively. CTCTT , (pdRib)3- pGGA-(pdRib)4-T and (pdRib)3- 50 30 pGGAT were synthesized by the standard phosphor- Preparation of [50-32P]-labelled RNA substrates amidite protocol on ASM-700 synthesizer (Biosset, Russia) using solid support, nucleoside phosphoramidites, A 96-nt fragment of RNA HIV-1 (RNA-96) was prepared dSpaser (pRib), chemical phosphorylation reagent by in vitro transcription using T7 RNA polymerase and from Glenn Research (USA). Oligonucleotides were Fok I—linearized plasmid pHIV-1 as described in (17). isolated by consecutive ion-exchange (Polysil SA-500 RNA-96 was dephosphorylated using bacterial alkaline columns, Russia) and reverse-phase HPLC (LiChrosorb phosphatase (BAP) according to described protocol (24). RP-18 columns, Merck, Germany) according to standard The reaction mixture, 50 ml of 50 mM Tris-HCl, pH 8.5, protocols.
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