Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7475-7480, July 1996 Biochemistry Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope (in vitro selection/SELEX/combining site) WEI XU AND ANDREW D. ELLINGTON* Department of Chemistry, Indiana University, Bloomington, IN 47405 Communicated by Norman R Pace, Indiana University, Bloomington, IN, April 3, 1996 (received for review December 1, 1995) ABSTRACT In vitro selection of nucleic acid binding been shown to interact with the anion exosite of the molecule species (aptamers) is superficially similar to the immune (5), whereas RNAs selected to bind to the Tax protein of response. Both processes produce biopolymers that can rec- human T-lymphotropic virus type I can recognize disparate ognize targets with high affinity and specificity. While anti- faces of the protein and differentially disrupt protein-protein bodies are known to recognize the sequence and conformation interactions with cellular transcription factors (6). of protein surface features (epitopes), very little is known We have employed the human immunodeficiency virus type about the precise interactions between aptamers and their 1 (HIV-1) Rev protein as a model system to further explore epitopes. Therefore, aptamers that could recognize a partic- how peptide and protein epitopes are recognized by aptamers. ular epitope, a peptide fragment of human immunodeficiency Rev binds to and facilitates the transport of mRNAs contain- virus type 1 Rev, were selected from a random sequence RNA ing a 234 residue Rev responsive element (RRE; ref. 7). pool. Several of the selected RNAs could bind the free peptide Interactions between the Rev protein and its primary binding more tightly than a natural RNA ligand, the Rev-binding site, the 30 residue Rev-binding element (RBE), have previ- element. In accord with the hypothesis that protein and ously been shown to be mediated by an arginine-rich motif nucleic acid binding cusps are functionally similar, interac- (ARM) that spans residues 34-50 (8, 9). In the current study, tions between aptamers and the peptide target could be RNA aptamers were selected to bind to the isolated Rev34_50 disrupted by sequence substitutions. Moreover, the aptamers peptide. Like antibodies, the selected RNAs could specifically appeared to be able to bind peptides with different solution recognize the sequence of the peptide. Like antibodies, the conformations, implying an induced fit mechanism for bind- anti-peptide aptamers could bind the corresponding native ing. Just as anti-peptide antibodies can sometimes recognize epitope on the Rev protein, albeit with lower affinity. These the corresponding epitope when presented in a protein, the results suggest that artificially selected binding cusps may have anti-peptide aptamers were found to specifically bind to Rev. much in common with those found on natural molecules and have important implications for the development of novel In vitro selection techniques have been used to isolate nucleic diagnostic reagents and strategies for intracellular immuniza- acid molecules that can bind tightly and specifically to a wide tion. range of target molecules (1). The sequence and functional diversity generated by in vitro genetic experiments is reminis- cent of the molecular diversity generated by an immune MATERIALS AND METHODS response. When the binding abilities of naturally selected Peptides, Proteins, and RNAs. Peptides were a gift of protein antibodies and artificially selected nucleic acid aptam- Amgen Biologicals. Peptides were made by standard auto- ers are compared, they are found to be strikingly similar. Like mated procedures. The purity and concentration of each antibodies, aptamers can recognize molecules as small as peptide was confirmed by HPLC, mass spectrometry, and theophylline (Mr = 120) and as large as 30S ribosomal particles amino acid analysis. The sequence of the 16-mer peptide (Mr > 106). Like antibody Fab fragments, aptamers can form corresponding to the ARM of Rex (positions 1-16) was monovalent complexes with their targets that have dissociation MPKTRRRPRRSQRKRP. The sequence of the 15-mer pep- constants in the nanomolar range. The specificity of aptamers tide corresponding to the ARM of Tat (positions 46-60) was for their targets can be similar to that of antibodies for their SYGRKKRRQRRRPPQ; this peptide was kindly provided by antigens: RNAs selected to bind to anti-peptide antibodies can Maria Zapp (University of Massachusetts Medical Center). compete with the cognate peptide ligand (2). RNAs selected Rev and a glutathione S-transferase (GST)-Rex fusion protein to bind to one isozyme of protein kinase C can discriminate were overexpressed in Escherichia coli and were generous gifts against isozymes that are as much as 96% homologous (3), of Maria Zapp. Recombinant Tat protein was overexpressed in while RNAs selected to bind to avian myeloblastosis virus E. coli and was obtained from Agmed (Bedford, MA). reverse transcriptase can discriminate against the Moloney A minimal RBE that binds Rev as well as the 94-nt stem IID murine leukemia virus reverse transcriptase enzyme and vice of the RRE was used for competition experiments. The versa (4). sequence of the minimal element was 5' GGGAACUCGAU- The hypothesis that the two types of biopolymers are GAAGCGAGCUCUUGGGCGCAGCCUCAAU- functionally similar can be further assessed by determining to GAGGCUGACGGUACAAGUACUGACUUCG- what extent the properties of antibodies predict the properties GAUCCCUGC; underlined positions correspond to residues of aptamers. Such predictions have already been borne out in 45-54 and 64-75 of the HIV-1 RRE [numbering according to several instances. For example, antibodies have long been Malim et al. (10)]. The minimal RBE was transcribed from a known to recognize particular surface features (epitopes) on double-stranded DNA oligomer by using an Ampliscribe kit antigens. Aptamers now also appear to recognize epitopes on target molecules. DNAs selected to bind to thrombin have Abbreviations: RRE, Rev responsive element; RBE, Rev binding element; ARM, arginine-rich motif; sRev, selection Rev; uRev, un- The publication costs of this article were defrayed in part by page charge modified Rev. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 7475 Downloaded by guest on October 2, 2021 7476 Biochemistry: Xu and Ellington Proc. Natl. Acad. Sci. USA 93 (1996) (Epicentre Technologies, Madison, WI) and was purified on a Competition Assays with the RBE. Aptamers were assayed denaturing polyacrylamide gel as described (11). for their ability to compete with the RBE for binding to Rev. Derivatization of Resin. A peptide corresponding to resi- Either radiolabeled N71 pool or selected clones (2.4 ,g; 0.6 dues 34-50 of the Rev protein of HIV-1 (sRev; see Fig. 4) was ,LM) were heated to 75°C for 3 min in 1 x binding buffer (50 used for selections. A milligram of peptide was coupled to 0.5 p,l) and allowed to cool to ambient temperature over S min. ml bed volume of Affi-gel 10 (Bio-Rad) according to the Radiolabeled RBE (1.8 ,ug; 0.6 ,uM; 50 ,l) was thermally manufacturer's instructions. The amount of peptide present in equilibrated in a separate tube. The two RNA samples were solution before and after the derivatization was determined by mixed and Rev protein was added to a final concentration of a Coomassie blue assay (12). The concentration of the peptide 0.3 ,M. The mixture was incubated at ambient temperature for on the resin was estimated to be 0.5 mg/ml (0.2 mM). A 60 min. A small portion (5 ,ul) of the reaction mixture was set "negative selection" resin was generated by derivatizing Affi- aside, and the remainder of the binding reaction was filtered gel 10 with ethanolamine. over HAWP modified cellulose filters (Millipore). The filters Pool Design and Construction. A RNA pool containing a were washed twice with 500 ,ll of lx binding buffer. Bound 71-nt completely random sequence tract was used as a starting RNAs were eluted by boiling three times in 100 ,ul water for point for selections (13). To avoid amplification artefacts, the 3 min, and the eluates were pooled (300 ,lI) and precipitated. primer sequences flanking the randomized region corre- Unfiltered and filtered samples were analyzed on a 10% sponded to those used by Crameri and Stemmer (14), with the denaturing polyacrylamide gel. The number of counts in exception that one of the primers (42.71), was appended to individual bands were determined by using a Phosphorlmager include T7 RNA polymerase promoter sequences. The se- (Molecular Dynamics). quence of 42.71 was 5' GGTAATACGACTCACTATAGG- GAGATACCAGCTTATTCAAT. A double-stranded DNA RESULTS pool was generated from the original synthetic oligomer via a large-scale PCR (13), and an RNA pool was transcribed from In Vitro Selection of Aptamers That Bind a Peptide from a portion (1 ,g; 1 x 1013 sequences) of this pool. HIV-1 Rev. The RNA recognition domain of HIV-1 Rev is an In Vitro Selection of Aptamers That Could Bind an Immo- ARM that spans residues 34-50. A 17-mer peptide (sRev) bilized Peptide. In round 1, RNA (5 gg, 5 library equivalents) corresponding to this domain was used as a target for in vitro in 1 x binding buffer (500 ,ul of 10 mM Hepes, pH 7.4/100 mM selection experiments. A RNA pool containing 71 random NaCl) was mixed with negative selection resin (50 ,ul) and sequence positions was incubated with an affinity resin con- 10 The binding reaction was poured taining sRev. Nonbinding species were removed by washing, incubated at 7°C for min. and binding species were eluted with high salt. The stringency into a small plastic tube, briefly centrifuged, and the super- of the selection was progressively increased by washing the natant removed.