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• W2038167103 abstract "Tenascin-C (TN-C) is an extracellular matrix protein that is overexpressed during tissue remodeling processes, including tumor growth. To identify an aptamer for testing as a tumor-selective ligand, SELEX (systematicevolution of ligands by exponential enrichment) procedures were performed using both TN-C and TN-C-expressing U251 glioblastoma cells. The different selection techniques yielded TN-C aptamers that are related in sequence. In addition, a crossover procedure that switched from tumor cell to purified protein selections was effective in isolating two high-affinity TN-C aptamers. When targeting tumor cells in vitro, the observed propensity of naive oligonucleotide pools to evolve TN-C aptamers may be due to the abundance of this protein.In vivo, TN-C abundance may also be well suited for aptamer accumulation in the tumor milieu. A size-minimized and nuclease-stabilized aptamer, TTA1, binds to the fibrinogen-like domain of TN-C with an equilibrium dissociation constant (Kd) of 5 × 10−9m. At 13 kDa, this aptamer is intermediate in size between peptides and single chain antibody fragments, both of which are superior to antibodies for tumor targeting because of their smaller size. TTA1 defines a new class of ligands that are intended for targeted delivery of radioisotopes or chemical agents to diseased tissues. Tenascin-C (TN-C) is an extracellular matrix protein that is overexpressed during tissue remodeling processes, including tumor growth. To identify an aptamer for testing as a tumor-selective ligand, SELEX (systematicevolution of ligands by exponential enrichment) procedures were performed using both TN-C and TN-C-expressing U251 glioblastoma cells. The different selection techniques yielded TN-C aptamers that are related in sequence. In addition, a crossover procedure that switched from tumor cell to purified protein selections was effective in isolating two high-affinity TN-C aptamers. When targeting tumor cells in vitro, the observed propensity of naive oligonucleotide pools to evolve TN-C aptamers may be due to the abundance of this protein.In vivo, TN-C abundance may also be well suited for aptamer accumulation in the tumor milieu. A size-minimized and nuclease-stabilized aptamer, TTA1, binds to the fibrinogen-like domain of TN-C with an equilibrium dissociation constant (Kd) of 5 × 10−9m. At 13 kDa, this aptamer is intermediate in size between peptides and single chain antibody fragments, both of which are superior to antibodies for tumor targeting because of their smaller size. TTA1 defines a new class of ligands that are intended for targeted delivery of radioisotopes or chemical agents to diseased tissues. extracellular matrix systematicevolution of ligands by exponential enrichment tenascin-C single-stranded DNA surface plasmon resonance reverse transcription-PCR Tenascin-C is a very large (>1 × 106 Da) hexameric glycoprotein that is located primarily in the extracellular matrix (ECM).1 TN-C is expressed during fetal development, wound healing, tumor growth, atherosclerosis and psoriasis, suggesting a role for this protein in tissue remodeling processes (reviewed in Refs. 1Erickson H.P. Bourdon M.A. Annu. Rev. Cell Biol. 1989; 5: 71-92Crossref PubMed Scopus (525) Google Scholar and 2Koukoulis G.K. Gould V.E. Bhattacharyya A. Gould J.E. Howeedy A.A. Virtanen I. Hum. Pathol. 1991; 22: 636-643Crossref PubMed Scopus (204) Google Scholar; see also Refs.3Mackie E.J. Halfter W. Liverani D. J. Cell Biol. 1988; 107: 2757-2767Crossref PubMed Scopus (469) Google Scholar, 4Wallner K. Li C. Shah P.K. Fishbein M.C. Forrester J.S. Kaul S. Sharifi B.G. Circulation. 1999; 99: 1284-1289Crossref PubMed Scopus (131) Google Scholar, 5Schalkwijk J. Steijlen P.M. van Vlijmen-Willems I.M. Oosterling B. Mackie E.J. Verstraeten A.A. Am. J. Pathol. 1991; 139: 1143-1150PubMed Google Scholar). As judged by Western blotting and immunohistochemical staining (6Hauptmann S. Zardi L. Siri A. Carnemolla B. Borsi L. Castellucci M. Klosterhalfen B. Hartung P. Weis J. Stocker G. et al.Lab. Invest. 1995; 73: 172-182PubMed Google Scholar, 7Riedl S. Kadmon M. Tandara A. Hinz U. Moller P. Herfarth C. Faissner A. Dis. Colon Rectum. 1998; 41: 86-92Crossref PubMed Scopus (19) Google Scholar, 8Sakai T. Kawakatsu H. Hirota N. Yokoyama T. Sakakura T. Saito M. Br. J. Cancer. 1993; 67: 1058-1064Crossref PubMed Scopus (53) Google Scholar, 9Zagzag D. Friedlander D.R. Miller D.C. Dosik J. Cangiarella J. Kostianovsky M. Cohen H. Grumet M. Greco M.A. Cancer Res. 1995; 55: 907-914PubMed Google Scholar, 10Lightner V.A. Slemp C.A. Erickson H.P. Ann. N. Y. Acad. Sci. 1990; 580: 260-275Crossref PubMed Scopus (30) Google Scholar, 11Borsi L. Carnemolla B. Nicolo G. Spina B. Tanara G. Zardi L. Int. J. Cancer. 1992; 52: 688-692Crossref PubMed Scopus (156) Google Scholar, 12Howeedy A.A. Virtanen I. Laitinen L. Gould N.S. Koukoulis G.K. Gould V.E. Lab. Invest. 1990; 63: 798-806PubMed Google Scholar, 13Mackie E.J. Chiquet-Ehrismann R. Pearson C.A. Inaguma Y. Taya K. Kawarada Y. Sakakura T. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4621-4625Crossref PubMed Scopus (235) Google Scholar, 14Soini Y. Paakko P. Nuorva K. Kamel D. Linnala A. Virtanen I. Lehto V.P. Am. J. Clin. Pathol. 1993; 100: 145-150Crossref PubMed Scopus (46) Google Scholar, 15Ibrahim S.N. Lightner V.A. Ventimiglia J.B. Ibrahim G.K. Walther P.J. Bigner D.D. Humphrey P.A. Hum. Pathol. 1993; 24: 982-989Crossref PubMed Scopus (49) Google Scholar, 16Xue Y. Li J. Latijnhouwers M.A. Smedts F. Umbas R. Aalders T.W. Debruyne F.M. De La Rosette J.J. Schalken J.A. Br. J. Urol. 1998; 81: 844-851Crossref PubMed Google Scholar), TN-C levels in tumors are significantly higher than in normal tissue. Further, TN-C levels are predictive of local tumor recurrence and are correlated with invasiveness and distant metastasis (17Jahkola T. Toivonen T. Nordling S. von Smitten K. Virtanen I. Eur. J. Cancer. 1998; 34: 1687-1692Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 18Ishihara A. Yoshida T. Tamaki H. Sakakura T. Clin. Cancer Res. 1995; 1: 1035-1041PubMed Google Scholar, 19Jahkola T. Toivonen T. Virtanen I. von Smitten K. Nordling S. von Boguslawski K. Haglund C. Nevanlinna H. Blomqvist C. Br. J. Cancer. 1998; 78: 1507-1513Crossref PubMed Scopus (106) Google Scholar), although these findings remain controversial. Tumor metastases can also express TN-C (10Lightner V.A. Slemp C.A. Erickson H.P. Ann. N. Y. Acad. Sci. 1990; 580: 260-275Crossref PubMed Scopus (30) Google Scholar, 20Dueck M. Riedl S. Hinz U. Tandara A. Moller P. Herfarth C. Faissner A. Int. J. Cancer. 1999; 82: 477-483Crossref PubMed Scopus (45) Google Scholar). In addition to localization in tumor stroma, TN-C can be associated with tumor vascular structures (21Vacca A. Ribatti D. Fanelli M. Costantino F. Nico B. Di Stefano R. Serio G. Dammacco F. Leuk. Lymphoma. 1996; 22: 473-481Crossref PubMed Scopus (33) Google Scholar, 22Zagzag D. Friedlander D.R. Dosik J. Chikramane S. Chan W. Greco M.A. Allen J.C. Dorovini-Zis K. Grumet M. Cancer Res. 1996; 56: 182-189PubMed Google Scholar, 23Kostianovsky M. Greco M.A. Cangiarella J. Zagzag D. Ultrastruct. Pathol. 1997; 21: 537-544Crossref PubMed Scopus (20) Google Scholar, 24Tokes A.M. Hortovanyi E. Kulka J. Jackel M. Kerenyi T. Kadar A. Pathol. Res. Pract. 1999; 195: 821-828Crossref PubMed Scopus (24) Google Scholar) and may promote angiogenesis through interaction with the integrin αvβ3 (25Yokoyama K. Erickson H.P. Ikeda Y. Takada Y. J. Biol. Chem. 2000; 275: 16891-16898Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Because of the abundance of TN-C in tumor stroma and its association with angiogenesis, high-affinity TN-C ligands may be clinically useful tumor-targeting agents. In fact, radiolabeled antibodies to TN-C are currently being evaluated in glioblastoma patients (26Bigner D.D. Brown M.T. Friedman A.H. Coleman R.E. Akabani G. Friedman H.S. Thorstad W.L. McLendon R.E. Bigner S.H. Zhao X.G. Pegram C.N. Wikstrand C.J. Herndon II, J.E. Vick N.A. Paleologos N. Cokgor I. Provenzale J.M. Zalutsky M.R. J. Clin. Oncol. 1998; 16: 2202-2212Crossref PubMed Scopus (163) Google Scholar, 27Riva P. Franceschi G. Frattarelli M. Lazzari S. Riva N. Giuliani G. Casi M. Sarti G. Guiducci G. Giorgetti G. Gentile R. Santimaria M. Jermann E. Maeke H.R. Clin. Cancer Res. 1999; 5: 3275s-3280sPubMed Google Scholar) with significant responses to treatment in a phase II study (28Riva P. Franceschi G. Frattarelli M. Riva N. Guiducci G. Cremonini A.M. Giuliani G. Casi M. Gentile R. Jekunen A.A. Kairemo K.J. Acta Oncol. 1999; 38: 351-359Crossref PubMed Scopus (73) Google Scholar). Aptamers are typified by high affinity and specificity for their cognate proteins (reviewed in Refs. 29Gold L. Polisky B. Uhlenbeck O. Yarus M. Annu. Rev. Biochem. 1995; 64: 763-797Crossref PubMed Scopus (735) Google Scholar, 30Famulok M. Mayer G. Curr. Top. Microbiol. Immunol. 1999; 243: 123-136PubMed Google Scholar, 31Osborne S.E. Matsumura I. Ellington A.D. Curr. Opin. Chem. Biol. 1997; 1: 5-9Crossref PubMed Google Scholar) and can be considered as oligonucleotide analogs of antibodies. However, as nucleic acids, aptamers are fundamentally distinct from antibodies. In having small size (8–15 kDa) relative to antibodies (150 kDa), aptamers are candidates for rapid tumor penetration and blood clearance. These are useful attributes for noninvasive diagnosis of disease (32Hicke B.J. Stephens A.W. J. Clin. Invest. 2000; 106: 923-928Crossref PubMed Google Scholar) and may provide advantages over antibodies and fragments thereof, which demonstrate slower tissue penetration and clearance rates. To identify an aptamer for investigation of tumor-targeting and blood clearance properties, we describe herein a SELEX process to identify TN-C aptamers and then focus attention on a single aptamer, TTA1. The SELEX process uses large (1014-1015sequences) oligonucleotide pools to identify binding species,i.e. aptamers, to a variety of purified molecular targets. In addition to generating aptamers against purified proteins/small molecules, SELEX technology can generate aptamers to cells (33Morris K.N. Jensen K.B. Julin C.M. Weil M. Gold L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2902-2907Crossref PubMed Scopus (280) Google Scholar) and tissues. 2A. Stephens, personal communication. 2A. Stephens, personal communication. The advantages of complex targets include freedom from the need to define and purify a molecular target, and presentation of proteins in native folding and glycosylation states. For complex SELEX experiments, identifying optimal selection conditions is theoretically possible (34Vant-Hull B. Payano-Baez A. Davis R.H. Gold L. J. Mol. Biol. 1998; 278: 579-597Crossref PubMed Scopus (72) Google Scholar) but remains a challenging task. In contrast, selection against purified protein allows ready experimental manipulation to achieve optimal enrichment of high-affinity aptamers (35Irvine D. Tuerk C. Gold L. J. Mol. Biol. 1991; 222: 739-761Crossref PubMed Scopus (201) Google Scholar) and requires no deconvolution to identify the cognate protein (or lipid, oligosaccharide, nucleic acid, etc.). Relative to cells and tissues, purified proteins often exhibit lower nonspecific binding of nucleic acids, and therefore selections proceed more rapidly. Because each has advantages, we elected to use both purified protein and cells as target sources to obtain TN-C ligands. A previous SELEX experiment targeting U251 glioblastoma cells identified a DNA aptamer that binds to tenascin-C, 3H. Chen and D. Daniels, personal communication. 3H. Chen and D. Daniels, personal communication. demonstrating that TN-C is a selectable target on U251 cells. The ssDNA aptamer displays greatly reduced binding affinity at physiological temperatures, 4B. Hicke, data not shown. 4B. Hicke, data not shown. perhaps because these initial cell SELEX experiments were performed at 4 °C. Thus the aptamer has relatively low affinity (Kd∼100 nm) at 37 °C and, being composed of DNA, is susceptible to nuclease activity in vivo. These features render the DNA aptamer unsuitable for in vivo applications. To identify aptamers for use as tumor-targeting agents, we undertook SELEX experiments at 37 °C using a nuclease-stabilized 2′-F pyrimidine oligonucleotide library. U251 cells, derived from a human glioblastoma, were obtained from the National Cancer Institute-Frederick Cancer Research Facility Tumor Repository and cultured in RPMI 1640 (Life Technologies, Inc.) + 10% fetal bovine serum (Summit, Ft. Collins, CO) to 90% confluency on tissue culture-treated polystyrene. Viability was checked using trypan blue staining. Human TN-C (Chemicon, Temecula, CA; other sources were inferior in purity and activity) was stored as frozen aliquots at −80 °C. After thawing, preparations could be stored for at least 2 months at 4 °C without loss of aptamer binding activity. Purified recombinant tenascin-C fragments (36Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar) were obtained from Harold P. Erickson, Duke University. Human and bovine serum albumin (fraction V) were obtained from Sigma. DNA oligonucleotides were obtained from Operon (Alameda, CA), and aptamer synthesis was performed at NeXstar Pharmaceuticals as described (37Wincott F. DiRenzo A. Shaffer C. Grimm S. Tracz D. Workman C. Sweedler D. Gonzalez C. Scaringe S. Usman N. Nucleic Acids Res. 1995; 23: 2677-2684Crossref PubMed Scopus (427) Google Scholar). Specialty phosphoramidites were obtained from Glen Research (Sterling, VA; (CH2CH2O)6 = Spacer 18; hexylamine; 3′dT polystyrene support), JBL Laboratories (San Luis Obispo, CA; 2′-F C and 2′-F U), or Proligo (Boulder, CO; PAC-protected 2′-OCH3-G, rG, and rA). 5′-amine-containing oligonucleotides were conjugated to succinimidyl biotin (Pierce) in 30% dimethyl formamide, 200 mm sodium borate, pH 9.0, at 25 °C for 15 min and purified on polyacrylamide gels. Alternatively, aptamer transcripts were 5′-biotinylated using a method described elsewhere. 5United States Patent No. 60/034,651 filed January 8, 1997. In brief, transcription of DNA templates is initiated with a 5-fold excess of a modified GMP over GTP in order to efficiently place a unique biotin on the 5′ end of the transcript. The modified GMP bears a hexyl amine moiety on the 5′ position that has been conjugated to biotin using the succinimidyl chemistry described above. These procedures were generally performed as described (38Fitzwater T. Polisky B. Methods Enzymol. 1996; 267: 275-301Crossref PubMed Scopus (149) Google Scholar). To prepare the initiating random library, double-stranded transcription templates were prepared by Klenow fragment extension of 40N7a ssDNA: 5′-TCGCGCGAGTCGTCTG(40N)CCGCATCGTCCTCCC-3′. The reverse complement of this sequence is the “sense” strand, representing the fixed sequences that span the random regions shown for individuals in Fig. 5. This was done using the 5N7 primer: 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′, which contains the T7 polymerase promoter (underlined). The32P-body labeled library was prepared with T7 RNA polymerase; all transcription reactions were performed in the presence of 2′-F pyrimidine nucleotides and 2′-OH purine nucleotides. For cell selections, U251 cells were grown to 90% confluence on 150-mm-diameter tissue culture plates and washed three times with 10 ml of binding buffer (Dulbecco's phosphate-buffered saline with MgCl2 and CaCl2 (Life Technologies, Inc.) and 0.05% bovine serum albumin). 1500 pmol of 32P body-labeled library was then incubated with the cells in 10 ml of binding buffer for 45 min with gentle shaking. Unbound oligonucleotides were removed using seven washes (10 min each) of 10-ml binding buffer, typically removing >99% of input radioactivity. A final 20–40-min wash of 5 ml included 10 mm EDTA that caused U251 cells to detach. Cells were pelleted (5 min at 300 × g) and the supernatant removed (the EDTA elution). Plates were then treated with 1 ml of Trizol (Life Technologies, Inc.), and the remaining cells/extracellular matrix were removed using a cell scraper. To form the final Trizol elution, pelleted cells from the EDTA elution were added to the Trizol extraction from the plate. After the first round of selection, the EDTA elution pool and Trizol elution pools were kept separate. In the Trizol arm, EDTA-sensitive aptamers were eluted and discarded before Trizol elution. All washes and incubations were at 37 °C in binding buffer. For EDTA elutions, the sample was extracted three times with a 1:1 mixture of phenol/chloroform and once with chloroform. To recover additional radioactivity, organic phases were back-extracted with 100 μl of 10 mm Tris, pH 7.5, and an additional volume of chloroform. Nucleic acid was then precipitated twice using 2 m NH4OH and 2.5 volumes of ethanol. For the Trizol elution, an additional 0.2 volume of chloroform was added to facilitate phase separation. The aqueous phase was then extracted twice with phenol:chloroform and once with chloroform and then treated with 5–10 μg of RNase A for 10 min at 37 °C to degrade contaminating cellular RNA (the aptamers are resistant to RNase A by virtue of 2′-F-modified pyrimidines). Organic phases were back-extracted as described for the EDTA elution. To precipitate nucleic acids, 0.25 volume of 0.8 m sodium citrate, 1.2m sodium chloride was added along with 0.25 volume of isopropanol. Reverse transcription (RT)-PCR and transcription were performed as described (38Fitzwater T. Polisky B. Methods Enzymol. 1996; 267: 275-301Crossref PubMed Scopus (149) Google Scholar). Two synthetic primers, 5N7 (see above) and 3N7a (5′-TCGCGCGAGTCGTCTG-3′), were used for RT-PCR. To monitor aptamer pool complexity, renaturation rates were measured as described (39Charlton J. Smith D. RNA (N. Y.). 1999; 5: 1326-1332Crossref PubMed Scopus (18) Google Scholar). These procedures were performed as described (40Drolet D.W. Jenison R.D. Smith D.E. Pratt D. Hicke B.J. Comb. Chem. High Throughput Screen. 1999; 2: 271-278PubMed Google Scholar). For each round, 96-well Lumino plates (Labsystems, Needham Heights, MA) were coated for 2 h at room temperature with 200 μl of Dulbecco's phosphate-buffered saline containing tenascin-C. Control wells lacked tenascin-C in this initial coating step. After being coated, the wells were blocked using HBSMC buffer (20 mm HEPES, pH 7.4, 137 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 1 g/liter human serum albumin) for rounds 1–5. For rounds 6–8, wells were blocked with HBSMC buffer containing 1 g/liter casein (I-block; Tropix). This switch of blocking agent was performed to decrease background binding of aptamer pools to the plate surface. Binding and wash buffer consisted of HBSMC buffer containing 0.1% Tween 20. The aptamer pool was diluted into 100 μl of binding buffer and incubated for 30 min at 37 °C in the protein-coated wells. After binding, six washes of 200 μl each were performed. The wells were then emptied and placed on top of a 95 °C heat block for 5 min (“heat elution”). Standard avian myeloblastosis virus (AMV)-reverse transcriptase reactions (50 μl) were performed at 48 °C directly in the well, and the reaction products were utilized for standard PCR and transcription reactions. Cloning and sequencing used standard procedures. To measure aptamer pool binding, U251 cells were grown to confluence in 12-well tissue culture plates (Falcon 3047, Becton Dickinson). After the cells were washed with binding buffer,32P body-labeled aptamer pools were incubated with cells in binding buffer for 40 min at 37 °C. Unbound radioactivity was removed by aspiration, and two 10-s washes were performed. Trizol was used to collect bound cpm, which were quantitated by liquid scintillation counting. For SPR, aptamer pools were 5′-biotinylated (described above) and immobilized to a streptavidin-containing surface (SA chip, BIACORE 2000, Biacore AB, Uppsala, Sweden) at a level of ∼1000 response units. Running buffer was HBSMC containing 0.005% Tween 20 (P20, Biacore AB). A reference flow cell for each experiment consisted of a random sequence oligonucleotide pool. Kinetic constants for TN-9 binding to TNfbg (the bacterially expressed fibrinogen-like domain) were determined using standard methods (41Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (266) Google Scholar, 42Karlsson R. Falt A. J. Immunol. Methods. 1997; 200: 121-133Crossref PubMed Scopus (489) Google Scholar). Nitrocellulose filter partitioning assays were performed as described (43Tuerk C. Gold L. Science. 1990; 249: 505-510Crossref PubMed Scopus (7692) Google Scholar). Briefly,32P end-labeled oligonucleotides at 0.5 × 10−10m were incubated with increasing concentrations of TN-C in HBSMC buffer + 0.01% (w/v) human serum albumin at 37 °C for 15 min. Reactions were then filtered over nitrocellulose, and bound cpm were quantitated. Data were fit to obtain binding constants as described (44Green L.S. Jellinek D. Jenison R. Ostman A. Heldin C.H. Janjic N. Biochemistry. 1996; 35: 14413-14424Crossref PubMed Scopus (362) Google Scholar). To measure binding of aptamers to protein immobilized on plates, anti-tenascin-C monoclonal antibodies (mTN12, mouse TN-specific, Sigma; HxBO6, human TN-specific, Harold Erickson, Duke University) were adsorbed to MicroLite-2 96-well plates (Dynex Technologies) in HBSMC buffer for 18 h at 4 °C. Wells were washed four times with HBSMC buffer and blocked using 200 μl of HBSMC buffer + 0.1% (v/v) I-block (Tropix) for 2 h at 22 °C. Tenascin-C was then captured by incubation with serum-free medium from cells expressing mouse TN-C (3t12, ATCC) or human TN-C (U251, described above) for 18 h at 4 °C. After six washes with 200 μl of HBSMC buffer containing 0.1% I-block and 0.05% Tween 20 (HBSMCIT buffer), 150 μl of 5′-biotinylated aptamer was incubated with each well for 30 min at 37 °C. This was followed by three washes of 10 s each (aptamers tend to have rapid on and off rates relative to antibodies) with HBSMCIT buffer, incubation with a 1:1000 dilution of streptavidin-alkaline phosphatase (Roche Molecular Biochemicals), and 3–5 washes in HBSMCIT. Bound aptamer/streptavidin-AP complexes were quantified by chemiluminescent detection using CSPD/Sapphire (Tropix/ABI) according to the manufacturer's instructions. Briefly, 700 μl of disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate, 1.2 ml of Sapphire were added to 10 ml of diethanolamine buffer, and 150 μl of the solution was added to each well. After 15 min at 22 °C in the dark, chemiluminescence was detected using a luminometer (LB 96P, Berthold, Nashua, NH). To identify aptamers to TN-C, a tripartite SELEX experiment was carried out as diagrammed in Fig. 1. In the first arm, purified TN-C was used. The second arm consisted of selection against a TN-C-expressing glioblastoma cell line, U251. This arm was subdivided into EDTA and Trizol “elutions” to recover bound aptamers. A third arm was a crossover from cell selections onto purified protein selections. Human TN-C was adsorbed to polystyrene 96-well microtiter plates. To initiate selections, a random aptamer pool consisting of 1014oligonucleotides was generated using RNA polymerase. The oligonucleotides contained 2′-F pyrimidines and 2′-OH purines with a 40-nucleotide random sequence region flanked by fixed sequences for RT-PCR. Selections were performed according to Drolet et al.(40Drolet D.W. Jenison R.D. Smith D.E. Pratt D. Hicke B.J. Comb. Chem. High Throughput Screen. 1999; 2: 271-278PubMed Google Scholar), essentially consisting of protein-oligonucleotide incubations, washes to remove unbound oligonucleotides, and RT-PCR amplification of the bound oligonucleotides. The amounts of protein with each well in a 96-well plate and the amount of input RNA are indicated in TableI.Table ITenascin-C, tumor cell, and crossover SELEX procedures: RNA and protein inputTenascin-C SELEX experimentRoundpmol protein/wellpmol RNA/well112 (6 wells)200 (6 wells)21220031220041220052336233723380.23.3Tumor cell SELEX experimentRoundPlate diameterpmol RNA/platemmE1/T12 × 1501500E2/T21501500E3/T31501500E4/T41501500E5/T51501500E6/T61501500E7/T71501500E8/T81501500E9/T91501500Crossover SELEX experiment: cells to Tenascin-CRoundpmol protein/wellpmol RNA/wellE9P1/T9P1233E9P2/T9P2233For the purified protein selections, protein input into each well represents the quantity of protein incubated with each well for adsorption, which was then incubated in buffer with the indicated quantity of RNA. Decreases in protein and RNA input occurred as the pool affinities improved. For the tumor cell SELEX experiment, U251 glioblastoma cells were grown to confluence in tissue culture plates for each round. Open table in a new tab For the purified protein selections, protein input into each well represents the quantity of protein incubated with each well for adsorption, which was then incubated in buffer with the indicated quantity of RNA. Decreases in protein and RNA input occurred as the pool affinities improved. For the tumor cell SELEX experiment, U251 glioblastoma cells were grown to confluence in tissue culture plates for each round. A qualitative assessment of PCR amplification indicated that background binding of the RNA pools to polystyrene without associated tenascin-C (“no protein” control) was increasing through the initial five rounds. At round 6, the blocking agent was switched from human serum albumin to casein, which resulted in dramatically decreased aptamer pool binding to the no protein control wells. Progress was quantitated by measuring the affinity of 32P-labeled aptamer pools for TN-C using a nitrocellulose filter capture assay (45Yarus M. Berg P. Anal. Biochem. 1970; 35: 450-465Crossref PubMed Scopus (129) Google Scholar). After five rounds, a slight improvement in binding was evident. Coincident with the switch in blocking agent, the amount of TN-C binding in the aptamer pool rose dramatically in round 6. By round 8, affinity had increased at least 1000-fold to an equilibrium dissociation constant (Kd) of 3 × 10−9m. As no further affinity improvement was evident in the subsequent round, selection was deemed complete at round 8. A second experiment used human U251 glioblastoma cells as the target source. These cells construct an ECM containing abundant TN-C (46Ventimiglia J.B. Wikstrand C.J. Ostrowski L.E. Bourdon M.A. Lightner V.A. Bigner D.D. J. Neuroimmunol. 1992; 36: 41-55Abstract Full Text PDF PubMed Scopus (76) Google Scholar). Cells were grown to confluence and incubated with 1014 sequences of a random oligonucleotide pool (identical to that described above) at 37 °C for 1 h. After extensive washing, a final wash buffer containing 10 mm EDTA was applied to elute EDTA-sensitive aptamers. Because nucleic acid structures and nucleic acid-protein interactions often utilize divalent cations, it was expected that EDTA would elute a subset of cell-bound aptamers. The cells were solubilized, nucleic acids were extracted using Trizol™, a reagent that combines chaotropic denaturation of proteins with organic extraction of nucleic acids, and then the remaining aptamers were collected. Thus the EDTA served to elute a subset of bound aptamers, and the subsequent Trizol elution collected all remaining aptamers along with cellular RNAs. Aptamers from both EDTA and Trizol elutions were amplified by RT-PCR and transcribed, closing the first round of this SELEX experiment. Unlike the purified protein SELEX experiment, cell and input RNA concentrations remained constant throughout nine rounds of selection (Table I). The progress of the cell selections was monitored by measuring the binding of radiolabeled aptamer pools to U251 cells. To analyze the EDTA elution SELEX experiment, Fig.2A compares binding of a control aptamer pool to rounds 3, 5, and 9. The control aptamer pool bound the cells detectably, and binding was saturable. Relative to this nonspecific binding, rounds 3, 5, and 9 showed progressively increasing binding. Similar to the EDTA elution pools, the Trizol pools showed increased binding compared with a random aptamer pool (Fig. 2B). The T9 (Trizol round 9) pool showed less apparent binding than the T5 pool. This was due to increased binding to the polystyrene surface (data not shown). This outcome suggests that Trizol-eluted aptamers bound to the polystyrene surface, directing selective pressure away from cell binding and toward polystyrene binding. The cell binding analysis demonstrated pool evolution toward U251 binding. However, this analysis did not fully evaluate the progression of the tumor cell SELEX experiment; this is because a pool of low-affinity ligands for an abundant protein would show higher cell binding than a pool of high-affinity ligands for a rare protein. For many applications, the latter pool is desirable. Therefore we employed another measure of progression, aptamer pool complexity, which can be estimated by measuring nucleic acid renaturation rates (Cot analysis) (39Charlton J. Smith D. RNA (N. Y.). 1999; 5: 1326-1332Crossref PubMed Scopus (18) Google Scholar). Decreasing pool complexity serves as a proxy for convergence upon a high-affinity solution. The Cot analysis predicted that the E9 pool would contain ∼100 different oligonucleotide sequences, whereas the T9 pool would contained ∼50,000 sequences (data not shown). Taken together, the cell binding andCot analyses indicated that EDTA was more effective than Trizol in driving pool convergence toward cell binding. To determine whether the U251 aptamer pools contain TN-C aptamers, binding was investigated using a surface plasmon resonance (SPR) assay. Aptamer pools were biotinylated at the 5′ terminus and immobilized, via streptavidin, onto the surface" @default.
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