- Email: [email protected]
An improved system for regenerative production of combinatorial RNA libraries
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 349 (2006) 312–314 www.elsevier.com/locate/yabio
Notes & Tips
An improved system for regenerative production of combinatorial RNA libraries Yu-Ju Tseng a, Feng-Yuan Yang b, Lo-Chun Au a,b,¤ a
b
Graduate Institute of Medical Technology, National Yang-Ming University, Taiwan, ROC Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan, ROC Received 4 October 2005 Available online 15 December 2005
Systematic evolution of ligands by exponential enrichment (SELEX)1 [1] is an oligonucleotide-based combinatorial selection process that has been used to isolate high-aYnity ligands termed aptamer [2] for proteins and low-M.W. targets [3–5]. In SELEX, DNA or RNA aptamers with the highest target aYnities are isolated from a very large pool of random sequence molecules by multiple rounds of binding, elution, and PCR ampliWcation. Singlestranded nucleic acids, which form stable three-dimensional structures, can be selected to bind a single dominant epitope of the target molecule. Aptamers may have dissociation constants at picomolar levels to their targets [6,7]. Moreover, a variety of chemical modiWcations can be introduced to nucleic acids, such as incorporation of radiolabels, Xuorescent probes, cross-linking reagents [8,9], and changes to the backbone or bases [10], adding functionality and stability to the nucleic acid structure. Aptamers are emerging as a class of molecules that may rival antibodies in both therapeutic and diagnostic applications. Since RNAs are richer in tertiary structure than singlestranded DNA, application of SELEX to a combinatorial RNA library, in general, provides a better chance of isolating desirable aptamers. However, SELEX with combinatorial RNA libraries is a laborious and complex process. It starts with PCR ampliWcation of a large population of single-stranded oligonucleotides containing random sequences in the middle [11,12]. The double-stranded DNAs formed, having a T7 promoter at the 5⬘ end, are then used for in vitro transcription to generate a combinatorial RNA library. The long oligonucleotide template needs to be *
Corresponding author. Fax: +886 2 28733024. E-mail address: [email protected] (L.-C. Au). 1 Abbreviation used: SELEX, systematic evolution of ligands by exponential enrichment. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.11.032
HPLC-puriWed to obtain full-length sequence and yield is always very low. It makes template DNA quite expensive. Unfortunately, the costly template DNA library needs to be removed by DNase I digestion before the RNA library can be used for the Wrst round of SELEX. In this report, we propose a system for direct generation of combinatorial RNA libraries without the PCR ampliWcation step; the costly template DNA library could be used repeatedly. As illustrated in Fig. 1, the lower strand is a template strand containing a N40-random sequence. The upper strand is a universal sequence containing the T7 promoter. The two strands anneal to form a double-stranded molecule only in the T7 promoter region, suYcient for in vitro transcription using the T7 RNA polymerase. This design circumvents the PCR ampliWcation step needed for the generation of large amounts of double-stranded template DNA. The upper strand (Fig. 1) also contains a biotin linked to the T7 promoter sequence via a hexameric dT linker and a base G at the 3⬘ end. T7 RNA polymerase transcription initiating from a G base is known to enhance yields of transcriptional products. Biotin serves to direct the DNA duplex to bind to streptavidin resin. In vitro transcription reaction is conducted with constant rotation to prevent precipitation of the resin/template DNA complex. After the reaction, the RNA products could easily be separated from the template DNA by centrifugation. To demonstrate eYcacy of the experimental design, 5 nmol each of an upper-strand sequence, biotin-TTTTTT TAATACGACTCACTATAG, and the lower strand, 5⬘-T CTAGATGTAGACGCACATA-N40-TTCTGGTCGGT CGACTCCTATAGTGAGTCGTATTA-3⬘ (T7 promoter is underlined), were annealed in 2.5 ml of a buVer containing 0.1 M NaCl, 20 mM Tris–HCl, pH 8.0, at 50 °C for 3 min. The template DNA solution was then mixed with a 0.45-ml slurry of streptavidin-linked resin (Catalog No.
Notes & Tips / Anal. Biochem. 349 (2006) 312–314
313
Fig. 1. A system for regenerative production of combinatorial RNA libraries. The template DNA is composed of upper and lower strands which form a double-stranded segment in the T7 promoter region. N40 represents a 40-nt sequence formed by equal incorporation of the 4 nucleotides at each position. The two hatched boxes Xanking N40 represent deWned sequences for annealing of the PCR primers. The biotin at the 5⬘ end of the upper strand leads to the binding of the template DNA to streptavidin resin for in vitro transcription of a combinatorial RNA library. After the reaction, RNA in the solution is separated from the template DNA/resin complex by centrifugation. The template DNA/resin complex pellet is stored for reusing.
53113; Pierce, Rockford, IL) and incubated at room temperature for 30 min. The template DNA/resin complex was pelleted by centrifugation. More than 75% of template DNA formed complexes with the streptavidin as measured by absorption at 260 nm for unbound DNA remaining in the solution (data not shown). The pellet was washed once with phosphate-buVered saline and resuspended in 1 ml buVer of the AmpliScribe T7 in vitro transcription kit (Catalog No. ASF3507; Epicentre, Madison, WI) containing 100 l T7 RNA polymerase. Reaction was carried out at 37 °C for 30 min with constant rotation. The RNA products in solution were separated from the template DNA/resin complex by a brief centrifugation. Twenty microliters of DNase I was added to the solution followed by incubation at 37 °C for 15 min (an optional step). RNA was precipitated by adding 125 l 3 M sodium acetate (pH 5.0), 50 l of 0.5 M EDTA, 3 ml of ethanol and standing at ¡20 °C for 3 h. RNA pellet was obtained by centrifugation and was rinsed once with chilled 70% alcohol. RNA was Wnally dissolved in 1 ml RNase-free H2O, and the RNAs product were visualized by polyacrylamide gel electrophoresis (Fig. 2A, lane 1). The major population of RNA in the RNA library is expected to be 78mer in size in reference to a tRNA size marker. Some smearing may result in sequence diversiWcation in the N40 region. The amount of RNA produced in solution was measured by absorption at 260 nm. We estimated that 48 nmol RNA was transcribed from 3.75 nmol of template DNA, indicating that one copy of the template could generate 13 RNA copies. To demonstrate that this RNA product could be used for SELEX, RT-PCR was performed using the Tth DNA polymerase according to the manufacturer’s protocol (Catalog No. M2101; Promega, Madison, WI). BrieXy, 2 l RNA in 2.5 l H2O was heated at 70 °C for 10 min. Five microliters of the reaction mixture was then prepared at as follows stated Wnal concentration: 1 mM MnCl2, 1X RT buVer, 1 mM dNTP, and 33 pmol 3⬘ primer (5⬘-TCTAGATGTAGACGC
Fig. 2. VeriWcation of the RNA library and the derived RT-PCR products. (A) Polyacrylamide gel electrophoresis of in vitro-transcribed RNA. RNA sample (2.5 l; see text) was analyzed by electrophoresis in a 16% denaturing polyacrylamide gel containing 8 M urea followed by ethidium bromide staining. Lanes 1–4, RNA samples derived from reuses of in vitro transcription (see text); lane 5, negative control, in vitro transcription without addition of the template; lane 6, RNA sample produced by PCRampliWed double-stranded template as described in conventional method [5]; lane 7, Escherichia coli tRNA (1.5 g), 81 mer on average, for use as a size reference. (B) Polyacrylamide gel electrophoresis of RT-PCR products generated with the described system. RT-PCR product (5 l; see text) was analyzed by electrophoresis in a 16% native polyacrylamide gel without adding urea followed by ethidium bromide staining. Lanes 1–4, RTPCR products generated from the use of RNA templates derived from reuses of in vitro transcription (see text); lane 5, negative control, RTPCR without addition of the RNA template; lane 6, PCR product from template DNA (the lower strand) that was ampliWed directly using the same 3⬘ and 5⬘ primers for use as a size reference of 95 bp.
ACATA-3⬘). The reaction mixture was kept at 54 °C for 2 min followed by the addition of 0.5 l of the Tth DNA polymerase (5 units) and heating at 70 °C for 10 min. Forty microliters of a PCR mixture, which contained 33 pmol each of 3⬘ and 5⬘ primers (5⬘-TAATACGACTCACTATA GGAGTCGACCGACCAGAA-3⬘), IX chelate buVer, and 2.5 mM MgCl2, was then added. The PCR program used was as follows: 95 °C, 2 min; 54 °C, 30 s; 70 °C, 30 s; 95 °C, 30 s for 15 cycles; and 70 °C, 4 min. The PCR products were analyzed by polyacrylamide gel electrophoresis (Fig. 2B, lane l).
314
Notes & Tips / Anal. Biochem. 349 (2006) 312–314
On the other hand, the pellet of the template DNA/resin complex was kept at 4 °C for 2-day intervals before the second and third reuses of in vitro transcription were conducted. The pellet was then stored at ¡20 °C for 1 week before the fourth reuse of in vitro transcription. The integrity of these RNA libraries and the RT-PCR product generated are shown in Figs. 2A and B, lanes 2, 3, and 4, respectively. The yields of the RNA libraries were in the 1.15–1.20 mg range. The results indicate that there was no apparent decline in quality when the system was reused several times. A control RNA produced by the conventional method is shown in Fig. 2A, lane 6. For the conventional method, the double-stranded template was initially generated by PCR ampliWcation of the whole single-stranded oligonucleotide library [5]. In this report, we have described a novel system in regenerative production of combinatorial RNA libraries, which we used directly and repeatedly for in vitro transcription. This system may be helpful to developing of RNA aptamers. References [1] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249 (1990) 505–510. [2] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bind speciWc ligands, Nature 346 (1990) 818–822. [3] L. Gold, Oligonucleotides as research, diagnostic, and therapeutic agents, J. Biol. Chem. 270 (1995) 13581–13584.
[4] S.M. Nimjee, C.P. Rusconi, B.A. Sullenger, Aptamers: an emerging class of therapeutics, Annu. Rev. Med. 56 (2005) 555–583. [5] S. Jeong, T.-Y. Eom, S.-J. Kim, S.-W. Lee, J. Yu, In vitro selection of the RNA aptamer against the Sialyl Lewis X and its inhibition of the cell adhesion, Biochem. Biophys. Res. Commun. 281 (2001) 237–243. [6] J. Ruckman, L.S. Green, J. Beeson, S. Waugh, W.L. Gillette, D.D. Henninger, L. Claesson-Welsh, N. Janjic, 2⬘-Fluoropyrimidine RNAbased aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGFinduced vascular permeability through interactions requiring the exon 7-encoded domain, J. Biol. Chem. 273 (1998) 20556–20567. [7] R.D. Jenison, S.D. Jennings, D.W. Walker, R.F. Bargatze, D. Parma, Derivation of RNA aptamer inhibitors of human complement C5, Immunopharmacology 42 (1998) 219–230. [8] A. Rhie, L. Kirby, N. Sayer, R. Wellesley, P. Disterer, L. Sylvester, A. Gill, J. Hope, W. James, A. Tahiri-Alaoui, Characterization of 2⬘Xuoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion, J. Biol. Chem. 278 (2003) 39697–39705. [9] K.N. Morris, K.B. Jensen, C.M. Julin, M. Weil, L. Gold, High aYnity ligands from in vitro selection: complex targets, Proc. Natl. Acad. Sci. USA 95 (1998) 2902–2907. [10] S. Hoshika, N. Minakawa, A. Matsuda, Synthesis and physical and physiological properties of 4⬘-thioRNA: application to post-modiWcation of RNA aptamer toward NF-kappaB, Nucleic Acids Res. 32 (2004) 3815–3825. [11] X.M. Zhang, N.S. Shao, M.G. Chi, M.J. Sun, Screening of RNA molecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichment, Acta Pharmacol. Sin. 24 (2003) 711–714. [12] H. Ulrich, M.H. Magdesian, M.J. Alves, W. Colli, In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion, J. Biol. Chem. 277 (2002) 20756–20762.
Notes & Tips
An improved system for regenerative production of combinatorial RNA libraries Yu-Ju Tseng a, Feng-Yuan Yang b, Lo-Chun Au a,b,¤ a
b
Graduate Institute of Medical Technology, National Yang-Ming University, Taiwan, ROC Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan, ROC Received 4 October 2005 Available online 15 December 2005
Systematic evolution of ligands by exponential enrichment (SELEX)1 [1] is an oligonucleotide-based combinatorial selection process that has been used to isolate high-aYnity ligands termed aptamer [2] for proteins and low-M.W. targets [3–5]. In SELEX, DNA or RNA aptamers with the highest target aYnities are isolated from a very large pool of random sequence molecules by multiple rounds of binding, elution, and PCR ampliWcation. Singlestranded nucleic acids, which form stable three-dimensional structures, can be selected to bind a single dominant epitope of the target molecule. Aptamers may have dissociation constants at picomolar levels to their targets [6,7]. Moreover, a variety of chemical modiWcations can be introduced to nucleic acids, such as incorporation of radiolabels, Xuorescent probes, cross-linking reagents [8,9], and changes to the backbone or bases [10], adding functionality and stability to the nucleic acid structure. Aptamers are emerging as a class of molecules that may rival antibodies in both therapeutic and diagnostic applications. Since RNAs are richer in tertiary structure than singlestranded DNA, application of SELEX to a combinatorial RNA library, in general, provides a better chance of isolating desirable aptamers. However, SELEX with combinatorial RNA libraries is a laborious and complex process. It starts with PCR ampliWcation of a large population of single-stranded oligonucleotides containing random sequences in the middle [11,12]. The double-stranded DNAs formed, having a T7 promoter at the 5⬘ end, are then used for in vitro transcription to generate a combinatorial RNA library. The long oligonucleotide template needs to be *
Corresponding author. Fax: +886 2 28733024. E-mail address: [email protected] (L.-C. Au). 1 Abbreviation used: SELEX, systematic evolution of ligands by exponential enrichment. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.11.032
HPLC-puriWed to obtain full-length sequence and yield is always very low. It makes template DNA quite expensive. Unfortunately, the costly template DNA library needs to be removed by DNase I digestion before the RNA library can be used for the Wrst round of SELEX. In this report, we propose a system for direct generation of combinatorial RNA libraries without the PCR ampliWcation step; the costly template DNA library could be used repeatedly. As illustrated in Fig. 1, the lower strand is a template strand containing a N40-random sequence. The upper strand is a universal sequence containing the T7 promoter. The two strands anneal to form a double-stranded molecule only in the T7 promoter region, suYcient for in vitro transcription using the T7 RNA polymerase. This design circumvents the PCR ampliWcation step needed for the generation of large amounts of double-stranded template DNA. The upper strand (Fig. 1) also contains a biotin linked to the T7 promoter sequence via a hexameric dT linker and a base G at the 3⬘ end. T7 RNA polymerase transcription initiating from a G base is known to enhance yields of transcriptional products. Biotin serves to direct the DNA duplex to bind to streptavidin resin. In vitro transcription reaction is conducted with constant rotation to prevent precipitation of the resin/template DNA complex. After the reaction, the RNA products could easily be separated from the template DNA by centrifugation. To demonstrate eYcacy of the experimental design, 5 nmol each of an upper-strand sequence, biotin-TTTTTT TAATACGACTCACTATAG, and the lower strand, 5⬘-T CTAGATGTAGACGCACATA-N40-TTCTGGTCGGT CGACTCCTATAGTGAGTCGTATTA-3⬘ (T7 promoter is underlined), were annealed in 2.5 ml of a buVer containing 0.1 M NaCl, 20 mM Tris–HCl, pH 8.0, at 50 °C for 3 min. The template DNA solution was then mixed with a 0.45-ml slurry of streptavidin-linked resin (Catalog No.
Notes & Tips / Anal. Biochem. 349 (2006) 312–314
313
Fig. 1. A system for regenerative production of combinatorial RNA libraries. The template DNA is composed of upper and lower strands which form a double-stranded segment in the T7 promoter region. N40 represents a 40-nt sequence formed by equal incorporation of the 4 nucleotides at each position. The two hatched boxes Xanking N40 represent deWned sequences for annealing of the PCR primers. The biotin at the 5⬘ end of the upper strand leads to the binding of the template DNA to streptavidin resin for in vitro transcription of a combinatorial RNA library. After the reaction, RNA in the solution is separated from the template DNA/resin complex by centrifugation. The template DNA/resin complex pellet is stored for reusing.
53113; Pierce, Rockford, IL) and incubated at room temperature for 30 min. The template DNA/resin complex was pelleted by centrifugation. More than 75% of template DNA formed complexes with the streptavidin as measured by absorption at 260 nm for unbound DNA remaining in the solution (data not shown). The pellet was washed once with phosphate-buVered saline and resuspended in 1 ml buVer of the AmpliScribe T7 in vitro transcription kit (Catalog No. ASF3507; Epicentre, Madison, WI) containing 100 l T7 RNA polymerase. Reaction was carried out at 37 °C for 30 min with constant rotation. The RNA products in solution were separated from the template DNA/resin complex by a brief centrifugation. Twenty microliters of DNase I was added to the solution followed by incubation at 37 °C for 15 min (an optional step). RNA was precipitated by adding 125 l 3 M sodium acetate (pH 5.0), 50 l of 0.5 M EDTA, 3 ml of ethanol and standing at ¡20 °C for 3 h. RNA pellet was obtained by centrifugation and was rinsed once with chilled 70% alcohol. RNA was Wnally dissolved in 1 ml RNase-free H2O, and the RNAs product were visualized by polyacrylamide gel electrophoresis (Fig. 2A, lane 1). The major population of RNA in the RNA library is expected to be 78mer in size in reference to a tRNA size marker. Some smearing may result in sequence diversiWcation in the N40 region. The amount of RNA produced in solution was measured by absorption at 260 nm. We estimated that 48 nmol RNA was transcribed from 3.75 nmol of template DNA, indicating that one copy of the template could generate 13 RNA copies. To demonstrate that this RNA product could be used for SELEX, RT-PCR was performed using the Tth DNA polymerase according to the manufacturer’s protocol (Catalog No. M2101; Promega, Madison, WI). BrieXy, 2 l RNA in 2.5 l H2O was heated at 70 °C for 10 min. Five microliters of the reaction mixture was then prepared at as follows stated Wnal concentration: 1 mM MnCl2, 1X RT buVer, 1 mM dNTP, and 33 pmol 3⬘ primer (5⬘-TCTAGATGTAGACGC
Fig. 2. VeriWcation of the RNA library and the derived RT-PCR products. (A) Polyacrylamide gel electrophoresis of in vitro-transcribed RNA. RNA sample (2.5 l; see text) was analyzed by electrophoresis in a 16% denaturing polyacrylamide gel containing 8 M urea followed by ethidium bromide staining. Lanes 1–4, RNA samples derived from reuses of in vitro transcription (see text); lane 5, negative control, in vitro transcription without addition of the template; lane 6, RNA sample produced by PCRampliWed double-stranded template as described in conventional method [5]; lane 7, Escherichia coli tRNA (1.5 g), 81 mer on average, for use as a size reference. (B) Polyacrylamide gel electrophoresis of RT-PCR products generated with the described system. RT-PCR product (5 l; see text) was analyzed by electrophoresis in a 16% native polyacrylamide gel without adding urea followed by ethidium bromide staining. Lanes 1–4, RTPCR products generated from the use of RNA templates derived from reuses of in vitro transcription (see text); lane 5, negative control, RTPCR without addition of the RNA template; lane 6, PCR product from template DNA (the lower strand) that was ampliWed directly using the same 3⬘ and 5⬘ primers for use as a size reference of 95 bp.
ACATA-3⬘). The reaction mixture was kept at 54 °C for 2 min followed by the addition of 0.5 l of the Tth DNA polymerase (5 units) and heating at 70 °C for 10 min. Forty microliters of a PCR mixture, which contained 33 pmol each of 3⬘ and 5⬘ primers (5⬘-TAATACGACTCACTATA GGAGTCGACCGACCAGAA-3⬘), IX chelate buVer, and 2.5 mM MgCl2, was then added. The PCR program used was as follows: 95 °C, 2 min; 54 °C, 30 s; 70 °C, 30 s; 95 °C, 30 s for 15 cycles; and 70 °C, 4 min. The PCR products were analyzed by polyacrylamide gel electrophoresis (Fig. 2B, lane l).
314
Notes & Tips / Anal. Biochem. 349 (2006) 312–314
On the other hand, the pellet of the template DNA/resin complex was kept at 4 °C for 2-day intervals before the second and third reuses of in vitro transcription were conducted. The pellet was then stored at ¡20 °C for 1 week before the fourth reuse of in vitro transcription. The integrity of these RNA libraries and the RT-PCR product generated are shown in Figs. 2A and B, lanes 2, 3, and 4, respectively. The yields of the RNA libraries were in the 1.15–1.20 mg range. The results indicate that there was no apparent decline in quality when the system was reused several times. A control RNA produced by the conventional method is shown in Fig. 2A, lane 6. For the conventional method, the double-stranded template was initially generated by PCR ampliWcation of the whole single-stranded oligonucleotide library [5]. In this report, we have described a novel system in regenerative production of combinatorial RNA libraries, which we used directly and repeatedly for in vitro transcription. This system may be helpful to developing of RNA aptamers. References [1] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249 (1990) 505–510. [2] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bind speciWc ligands, Nature 346 (1990) 818–822. [3] L. Gold, Oligonucleotides as research, diagnostic, and therapeutic agents, J. Biol. Chem. 270 (1995) 13581–13584.
[4] S.M. Nimjee, C.P. Rusconi, B.A. Sullenger, Aptamers: an emerging class of therapeutics, Annu. Rev. Med. 56 (2005) 555–583. [5] S. Jeong, T.-Y. Eom, S.-J. Kim, S.-W. Lee, J. Yu, In vitro selection of the RNA aptamer against the Sialyl Lewis X and its inhibition of the cell adhesion, Biochem. Biophys. Res. Commun. 281 (2001) 237–243. [6] J. Ruckman, L.S. Green, J. Beeson, S. Waugh, W.L. Gillette, D.D. Henninger, L. Claesson-Welsh, N. Janjic, 2⬘-Fluoropyrimidine RNAbased aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGFinduced vascular permeability through interactions requiring the exon 7-encoded domain, J. Biol. Chem. 273 (1998) 20556–20567. [7] R.D. Jenison, S.D. Jennings, D.W. Walker, R.F. Bargatze, D. Parma, Derivation of RNA aptamer inhibitors of human complement C5, Immunopharmacology 42 (1998) 219–230. [8] A. Rhie, L. Kirby, N. Sayer, R. Wellesley, P. Disterer, L. Sylvester, A. Gill, J. Hope, W. James, A. Tahiri-Alaoui, Characterization of 2⬘Xuoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion, J. Biol. Chem. 278 (2003) 39697–39705. [9] K.N. Morris, K.B. Jensen, C.M. Julin, M. Weil, L. Gold, High aYnity ligands from in vitro selection: complex targets, Proc. Natl. Acad. Sci. USA 95 (1998) 2902–2907. [10] S. Hoshika, N. Minakawa, A. Matsuda, Synthesis and physical and physiological properties of 4⬘-thioRNA: application to post-modiWcation of RNA aptamer toward NF-kappaB, Nucleic Acids Res. 32 (2004) 3815–3825. [11] X.M. Zhang, N.S. Shao, M.G. Chi, M.J. Sun, Screening of RNA molecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichment, Acta Pharmacol. Sin. 24 (2003) 711–714. [12] H. Ulrich, M.H. Magdesian, M.J. Alves, W. Colli, In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion, J. Biol. Chem. 277 (2002) 20756–20762.