RNA Aptamer against Autoantibodies Associated with Multiple

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RNA Aptamer against Autoantibodies Associated with Multiple Sclerosis and Bioluminescent Detection Probe on Its Basis Maria A. Vorobjeva,† Vasilisa V. Krasitskaya,‡,§ Alesya A. Fokina,† Valentina V. Timoshenko,† Georgy A. Nevinsky,† Alya G. Venyaminova,† and Ludmila A. Frank*,‡,§ †

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk 630090, Russia ‡ Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk 660036, Russia § Siberian Federal University, Krasnoyarsk 660041, Russia S Supporting Information *

ABSTRACT: Nowadays, there are no specific laboratory tests for establishing the diagnosis of multiple sclerosis (MS). The presence of proteolytic autoantibodies against myelin basic protein is now considered as a characteristic feature of MS. New 2′-Fcontaining RNA aptamer of high affinity and specificity to these antibodies was selected. Covalent conjugate of this aptamer and Ca2+-regulated photoprotein obelin was obtained for the first time and applied as a label in bioluminescent microplate assay to detect target antibodies. The developed model solid-phase microassay is simple, fast, and highly sensitive.

M

The aim of our research was to select novel high-affinity RNA aptamers against MS-related autoantibodies and to develop aptamer-based sensitive and specific microplate assay to detect these autoantibodies in biological samples. Ca2+regulated photoprotein obelin was taken as a highly sensitive bioluminescent reporter.8 Covalent conjugate of new 57-nt 2′F-RNA aptamer and obelin was obtained and applied as a label in solid-phase bioluminescent assay of the pathological autoantibodies.

ultiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease of the human central nervous system. It is characterized by the progressive neurodegeneration leading to long-term disability and most commonly affects young adults.1 To verify the MS diagnosis, a combination of diagnostic tests is necessary, including magnetic resonance imaging and cerebrospinal fluid analysis,2 which are expensive and time-consuming. Nowadays, there are no specific laboratory tests for establishing the diagnosis of MS. During the past decade, myelin-specific autoantibodies, particularly antibodies against myelin basic protein (MBP), were discovered to contribute to the development of demyelination and disease progression due to their proteolytic activity.3,4 The presence of proteolytic anti-MBP autoantibodies is now considered to be a characteristic feature of MS.5 We supposed that RNA aptamers capable of specific recognition of these autoantibodies could be a basis for designing biosensors for MS detection. Aptamers are now widely used as recognizing elements in creation of biosensors (aptasensors) for specific and sensitive detection of a large variety of analytes from metal ions to proteins,6 with aptasensors for clinical diagnostics being of special interest among them.7 © 2014 American Chemical Society

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EXPERIMENTAL SECTION

Chemicals and Reagents. Polyclonal anti-MBP IgG, IgM, and IgA autoantibodies from the blood plasma of 12 MS patients (16−55 years old; men and women) with clinically definite MS according to the Poser criteria9 and total IgGs from the plasma of 12 healthy donors were purified as described in refs 4 and 10. The blood sampling protocol was applied in a city clinic in conformity with the local human ethics committee Received: November 22, 2013 Accepted: February 11, 2014 Published: February 11, 2014 2590

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Figure 1. SELEX of 2′-F-Py RNA aptamers against anti-MBP autoantibodies from MS patients, synthesis of the aptamer-obelin conjugate Apt2− 9c∼Obe (A) and its properties: bioluminescent signal of obelin (black) and the conjugate (red) triggered with Ca2+ (B); dependence of bioluminescence on conjugate concentration (C); absorption spectrum (D).

MBP IgGs were adsorbed on the walls of a polymerase chain reaction (PCR) tube, as described in ref 13. The 2′-F-Py RNA library (1 nmol in 100 μL of the binding buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM KCl) was preincubated in nonmodified PCR tube and then incubated in IgG-containing tube for 1 h at 25 °C. Afterward, all nonbound RNAs were discarded and the tube was washed with 500 μL of the buffer (50 mM Tris-HCl, pH 7.5, 200 mM KCl, 0.05% Tween 20). The remaining IgG-bound RNAs were heated for 2 min at 90 °C to denature RNA−protein complexes and subjected to reverse transcription in the same tube. The resulted cDNA was then PCR-amplified and the product was used as a template for RNA transcription. The pool of 2′-F-Py RNA molecules was gel-purified and used in the next round of selection. The selection pressure was increased progressively, reducing the time of incubation with the target antibodies from 1 h for the first six rounds to 30 min on the 10th round. After 10 rounds, the enriched library was cloned in Escherichia coli and 45 clones were sequenced using ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in SB RAS Genomics Core Facility. A series of 2′-F-Py RNA aptamers for primary screening of binding affinity after SELEX was synthesized by in vitro transcription (for aptamer sequences, see Table S-1 in the Supporting Information). Binding Assays. (A) The nitrocellulose filter binding assay is described in the Supporting Information, S-3. (B) For the gel-shift binding assay, the trace amounts of 3′-[32P]-labeled

guidelines (Ethics Committee of Novosibirsk State Medical University, Novosibirsk, Russia; Institutional Ethics Committee specifically approved this study in accordance with Helsinki Ethics Committee Guidelines). Monoclonal mouse IgG against hIgG Fc fragment (clone X53) was from Biosan (Russia). 2′-Fluoropyrimidine nucleotide triphosphates (2′-F-CTP, 2′F-UTP) were from Nanotex-C (Russia). The initial ssDNA library, ssDNA templates for 2′-fluoro pyrimidine nucleotide-containing (2′-F-Py) RNA aptamers, DNA primers, and truncated 2′-F-Py RNA aptamers (Apt2−9a, Apt2−9b, and Apt2−9c) were synthesized on the automatic ASM-800 RNA/DNA synthesizer (Biosset, Russia) using UltraMild β-cyanoethyl phosphoramidites and corresponding polymer supports (Glen Research, USA) according to protocols optimized for this instrument, deprotected using standard procedures, and purified by denaturing PAGE. The highly purified Ca2+-regulated photoprotein obelin with the unique cysteine residue (Obe-SH) was obtained as previously described.11 Solid-phase immunoassay was carried out using the 96-well opaque microtiter plates (Costar, Corning Inc., Corning, NY, USA). SELEX Procedure. A combinatorial 71-nt library of 2′-F-Py RNA containing a central stretch of 40 randomized nucleotides was obtained according to ref 12 using a synthetic ssDNA template library (∼1014 individual sequences). Polyclonal anti2591

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RESULTS AND DISCUSSION To recognize specifically MS-related proteolytic autoantibodies, new RNA aptamers were selected (Figure 1A). As a rule, monoclonal antibodies are used as targets for aptamers’ selection in similar cases (see, e.g., ref 14). We suppose that selection against the mixture of polyclonal anti-MBP IgGs from different patients would increase the possibility to obtain an aptamer binding to epitopes that are common for all target antibodies. The SELEX protocol developed by Missalidis13 for DNA aptamers was adapted in this study to the selection of RNA aptamers. RNA was chosen because of its ability to form a wider variety of 3D structures as compared to DNA because of the presence of 2′-OH groups. All pyrimidine nucleotides throughout the RNA library were replaced by their 2′-fluoro analogs to enhance aptamers’ nuclease resistance. For each round, polyclonal anti-MBP IgGs from the blood plasma of MS patients were adsorbed on the walls of the PCR tube and the stages of selection, washing of nonbound RNAs, and reverse transcription of bound RNAs were performed in the same tube. After 10 rounds of selection, an enriched 2′-F-Py RNA library was cloned and 45 clones were sequenced. The aptamers appearing more than once in the resulting set were further screened for the binding affinity toward pathogenic polyclonal IgGs using nitrocellulose filter binding assay (Table S-2, Supporting Information). Aptamer Apt2−9 possessed the highest affinity (the lowest Kd value) similar to that of the enriched 2′-F-Py RNA library; thus, it was chosen for subsequent studies. The secondary structure for Apt2−9 (of 71-nt length) obtained using Mfold structure predicting algorithm15 turned out to be a stem loop with a relatively long unpaired 3′fragment. We proposed that this fragment is dispensable for the high affinity binding to target autoantibodies. To prove this suggestion, three aptamers with 5, 10, and 14-nt 3′-end deletions (Apt2−9a, Apt2−9b, and Apt2−9c, correspondingly) were chemically synthesized and their affinities were estimated. In this case the mixture of polyclonal anti-MBP IgG + IgA + IgM from the blood plasma of MS patients was used as a target to evaluate the ability of the designed aptamers to bind with all anti-MBP MS-related immunoglobulins. The affinity was assayed using nondenaturing PAGE. As it can be seen from Kd values (Table 1), all 3′-end deletions resulted in the increase

aptamers and 0.5−500 nM of anti-MBP autoantibodies or IgGs from healthy donors were incubated in the binding buffer overnight at 25 °C, and then the reaction mixtures were analyzed by 6% nondenaturing PAGE. For quantitative data, the gels were analyzed by Molecular Imager FX (Bio-Rad Laboratories Inc., CA, USA). Equilibrium dissociation constants (Kd) were determined from binding curves using the GraphPad Prism 5.0.4.533 program package. Synthesis of Aptamer∼Obelin Conjugate Apt2− 9c∼Obe. The truncated 57-nt 2′-F-Py RNA aptamer Apt2− 9c∼NH2 containing aminopropyl linker at the 3′-end, was synthesized by the solid-phase phosphoroamidite synthesis as described above, using 3′-PT-amino-modifier C3 CPG polymer support (Glen Research, Sterling, VA, USA). The structure of Apt2−9c∼NH2 was confirmed by MALDI-TOF mass spectrometry. Apt2−9c∼NH2 was modified by the 50-fold molar excess of succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) (Fluka, Switzerland) for 1.5−2 h at room temperature in 0.1 M 2-(Bis(2-hydroxyethyl)amino)acetic acid (BICINE), pH 8.5. The reagent excess was removed on a DSalt Desalted Column (Pierce, Rockford, IL, USA) equilibrated with 20 mM Tris-HCl, pH 7.0, 5 mM ethylenediaminetetraacetic acid (EDTA). The SMCC-activated Apt2−9c∼NH2 was incubated with the 3-fold molar excess of Obe-SH overnight at 8 °C. The conjugate obtained was purified by anion-exchange chromatography on Mono Q column (GE Healthcare, BioSciences AB, Uppsala, Sweden), equilibrated with 20 mM TrisHCl, pH 7.0, 5 mM EDTA in NaCl concentration gradient (0− 2 M). Conjugate Apt2−9c∼Obe Detection Limit. A stock solution of the conjugate Apt2−9c∼Obe (1.2 × 10−11 M) was serially diluted in the assay buffer (20 mM Tris-HCl, pH 7.0, containing 5 mM EDTA and 0.1% bovine serum albumin) and placed into the wells (50 μL of each dilution), and bioluminescence intensity was measured with a plate luminometer LB 940 Multimode Reader Mithras (Berthold, Germany) immediately after rapid injection of CaCl2 solution (50 μL, 0.1 M in 0.1 M Tris-HCl, pH 8.8). The light was integrated for 5 s. The signal from the assay buffer (50 μL) was taken as a background. All measurements were performed in three replicates. Detection limit was determined as Apt2− 9c∼Obe quantity with a signal-to-background ratio of 3. Bioluminescent Solid-Phase Assay. The surface of microtiter wells was activated with 50 μL of monoclonal mouse IgG against hIgG Fc fragment (clone X-53) 10 μg/mL solutions in phosphate buffered saline (PBS) (0.15 M NaCl, 50 mM K−Na phosphate buffer, pH 7.0) overnight at 8 °C, then washed (three times, PBS, 0.1% Tween 20, 5 mM EDTA), and blocked with 1% fat-free milk powder in PBS (100 μL/well), 1 h, at 20 °C. After washing, 50 μL of anti-MBP autoantibodies or IgGs from healthy donor aliquots (concentrations in both cases ranged from 400 to 0.39 nM in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) were placed into the wells, incubated with shaking for 1 h at 20 °C, and washed thereafter. Then 50 μL of conjugate Apt2−9c∼Obe solution (21 ng/mL in 50 mM Tris-HCl, pH 7.5, 200 mM KCl, 2 mM EDTA) were placed into the wells, incubated at room temperature for 40 min, and then washed. Bioluminescence of the conjugate immobilized on the surface was measured as described above.

Table 1. Equilibrium Dissociation Constants (Kd) of 2′-F-Py RNA Aptamer−Antibody Complexes Kd, nM 2′-F-Py RNA Apt2−9 Apt2−9a Apt2−9b Apt2−9c a

anti-MBP IgG + IgA + IgM, MS patients

IgG, healthy donors

± ± ± ±

425 n.d.a n.d.a 2300 ± 460

15 3.0 1.4 1.2

4 0.5 0.7 0.1

Aptamer−antibody complexes were not detected.

of binding affinity. We suppose that the deletion of the 3′-end aptamer fragment decreases the probability of the formation of less stable alternative RNA structures. This increases the fraction of aptamers with the most stable secondary structure with high binding affinity. The binding of total IgG from healthy donors was also evaluated as a nonspecific control (Table 1). Full-length aptamer Apt2−9 possesses a pronounced specificity of binding (28-fold Kd difference for specific 2592

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and nonspecific binding). The 3′-shortened variants of the aptamer demonstrate even higher specificity: almost 2000-fold Kd difference was shown for Apt 2−9c, and in the case of Apt2−9b and Apt2−9a no aptamer−protein complexes were observed for IgGs from healthy donors, so we could not obtain Kd values. Therefore, the most shortened aptamer Apt2−9c possessing high affinity and specificity to the target antibodies was chosen for the following investigations. Since the 3′-end of the aptamer is not crucial for its binding ability, an aliphatic aminopropyl linker −(CH2)3NH2 was introduced to it in Apt2−9c during solid-phase chemical synthesis. To produce a label, this aptamer was covalently conjugated with Ca2+-regulated photoprotein obelin. This photoprotein as well as the other ones (aequorin, clytin, etc.) is a stable complex of a single-chain polypeptide (22.2 kDa) and 2-hydroperoxycoelenterazine (oxygen “preactivated” substrate).16 Nothing but calcium ions are required to trigger photoprotein’s bioluminescence. Because of the high quantum yield and low-level background, obelin may be determined in attomolar quantity that makes it an excellent reporter molecule.8 For conjugation with aptamer carrying aliphatic amino group, we used the mutant variant of obelin with the unique cysteine residue at its flexible N-terminus exposed to solution (Obe-SH). Commercially available heterobifunctional reagent succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1carboxylate (SMCC), reactive toward amino and sulfhydryl groups, was applied for Apt2−9c and obelin coupling. The loss of obelin bioluminescent activity at the conjugation was negligible (about 10−15%). The resulting conjugate possessed calcium triggering bioluminescence close to that of wild-type obelin (Figure 1B). The relationship between signal intensity and conjugate concentration is linear over several log units because of the nature of the photoprotein (Figure 1C). The conjugate detection limit is 2.5 amol (with a signal-tobackground ratio of 3, three replicates), close to the one for a wild-type obelin (1.5−2 amol). The absorption spectrum (Figure 1D) reveals a maximum at 260 nm, belonging to 2′-FPy RNA aptamer module with molar extinction 5.377 × 105 M−1 cm−1 (calculated17). The obelin module absorption at 280 nm with essentially lower molar extinction of 5.55 × 104 M−1 cm−1 makes only a minor contribution to the conjugate’s absorption spectrum. The conjugate was examined as a bioluminescent reporter in solid-phase assay according to the scheme, presented in Figure 2. The surface of the wells was activated with monoclonal mouse IgG against human IgG Fc fragment. The aliquots of target anti-MBP autoantibodies were placed into the wells and incubated; thereafter, the unbound targets were washed out. Then the solution of Apt2−9c∼Obe was placed into the wells, incubated, and washed. The IgG aliquots from healthy donors were used as the assay control. Bioluminescence of the label immobilized on the surface was triggered with Ca2+ injection and measured immediately. Figure 2 demonstrates the obtained results: all immunoglobuline samples from healthy donors give approximately equal low signal, whereas signals from samples of target anti-MBP autoantibodies depend on autoantibody concentration. Equilibrium dissociation constants (Kd) were determined from the binding curve using the Sigma Plot 2000 program package. Calculated dissociation constant (Kd) of the complex of target anti-MBP autoantibodies and Apt2−9c∼Obe is 8.2 ± 0.5 nM. At the target autoantibody concentration of 1.56 nM, the bioluminescent signal exceeds the control one 2.2 times. This concentration could be regarded as the assay sensitivity. The

Figure 2. Bioluminescent solid-phase microassay of the target antiMBP autoantibodies from MS patients (circuses) and IgG from healthy donors (triangles). Black and red circuses correspond to the results obtained with freshly prepared and frozen−melted conjugates. Each point is an average ±1 standard deviation (n = 3).

conjugate has not been inactivated under a frozen−melting procedure (Figure 2, red circuses) and after storage at 8 °C at least for 3 weeks. Thus, the obtained conjugate is a stable bifunctional molecule applicable as a label in bioluminescent solid-phase assay. The advantages of this label as compared to traditional antibody−enzyme conjugates are conditioned by the 2′-F-Py RNA aptamer as a recognition element: it is small in size, chemically stable, and once selected, can be obtained by chemical synthesis with high reproducibility and purity. Obelin as a reporter provides high sensitivity of the assay. When conjugated, both aptamer and obelin retain their properties and function properly. The developed model solid-phase microassay is simple, fast, and highly sensitive.

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CONCLUSIONS In this study, the novel 2′-F-Py RNA aptamer with high affinity and specificity to proteolytic anti-MBP autoantibodies from MS patients was selected and conjugated covalently with bioluminescent reporter, Ca2+-regulated photoprotein obelin. The new bifunctional conjugate was successfully applied as a label in model solid-phase assay of the pathologic anti-MBP autoantibodies. Positive experimental results demonstrate the proof-ofprinciple for detection of MS-specific autoantibodies using bioluminescent aptamer conjugates and provide the background to the next step of our investigationthe development of the laboratory tests for establishing the diagnosis of MS.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+7-391) 2494430. Fax: (+7-391) 2433400. Notes

The authors declare no competing financial interest. 2593

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ACKNOWLEDGMENTS The work was supported by RFBR grants No. 11-04-01014 and 14-04-01611, by Grant of the RF Government for support to scientific research projects implemented under the supervision of the world’s leading scientists, agreement No. 14.B25.31.0028, and by the SB RAS within the framework of the Interdisciplinary Integration Project No. 139. The authors are grateful to Anna Timofeyeva (ICBFM SB RAS) for the purification of antibodies from blood samples, to Anastasiya Popovetskaya for participating in SELEX experiments, and to Dr. Dmitry Zharkov for helping with aptamer cloning and sequencing experiments.

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REFERENCES

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