The use of mRNA display to select high-affinity protein-binding peptides - PubMed (original) (raw)

The use of mRNA display to select high-affinity protein-binding peptides

D S Wilson et al. Proc Natl Acad Sci U S A. 2001.

Abstract

We report the use of "mRNA display," an in vitro selection technique, to identify peptide aptamers to a protein target. mRNA display allows for the preparation of polypeptide libraries with far greater complexity than is possible with phage display. Starting with a library of approximately 10(13) random peptides, 20 different aptamers to streptavidin were obtained, with dissociation constants as low as 5 nM. These aptamers function without the aid of disulfide bridges or engineered scaffolds, yet possess affinities comparable to those for monoclonal antibody-antigen complexes. The aptamers bind streptavidin with three to four orders of magnitude higher affinity than those isolated previously by phage display from lower complexity libraries of shorter random peptides. Like previously isolated peptides, they contain an HPQ consensus motif. This study shows that, given sufficient length and diversity, high-affinity aptamers can be obtained even from random nonconstrained peptide libraries. By engineering structural constraints into these ultrahigh complexity peptide libraries, it may be possible to produce binding agents with subnanomolar binding constants.

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Figures

Figure 1

Figure 1

In vitro selection process. Schematic showing the structure of the library and the selection scheme. The DNA library has, from 5′ to 3′, a T7 RNA polymerase promoter (T7), a tobacco mosaic virus translation enhancer (TMV; ref. 36), a start codon (ATG), 88 random amino acids, a hexahistidine tag (H6), and a 3′ constant region (Const). This library is transcribed by using T7 RNA polymerase, after which the puromycin-containing linker is ligated onto the 3′ end of the mRNA. When this template is translated in vitro, the nascent peptide forms a covalent bond to the puromycin moiety. The resulting mRNA-peptide covalent fusion molecules are then purified on oligo-dT-cellulose (which anneals to the oligo-dA sequence in the puromycin-containing linker) and Ni-NTA agarose. The mRNA portion of this display construct is then reverse transcribed. The double-stranded DNA/RNA-peptide species are then incubated with the immobilized target protein (SA) and unbound library members are washed off. SA-bound peptides are then displaced with biotin. The eluted molecules are then amplified by PCR, thus completing the first round of selection and amplification.

Figure 2

Figure 2

Progress and result of the selection. (A) Fraction of35S counts from the displayed peptides that bound to SA and eluted with biotin, at each round of selection. (B) Elution profile for the peptide library generated from the output of the seventh round of selection. The first fraction represents the flow-through. Biotin was added at the point indicated. The plot compares the binding of the intact reverse-transcribed displayed peptides (mRNA-pep), the same sample treated with RNase A, and the RNase-treated sample applied to an SA column presaturated with biotin (excess biotin was washed away before exposing the library to the matrix).

Figure 3

Figure 3

EMSA analysis of peptide-SA interactions. (A) Qualitative demonstration of the binding of four different DNA-tagged peptides to SA. The migration of each clone is shown in the absence (−) and presence (+) of 1 μM SA. Some of the clones show multiple bands, presumably representing different conformations. The arrows show the position of the gel well, which often contains a fraction of the counts. (B) Titration of clone SB19 (full-length) with SA. The SA concentration in each lane, from left to right, is: 3.8, 6.6, 10, 15, 23, 35, and 61 nM. (C) Curve fitted to the data shown in B (the fraction of peptide bound could not be accurately determined for the point with the lowest concentration of SA). Assuming that the peptide is homogeneous and 100% active, the data from this experiment give a _K_D of 11 nM (_K_D = 10 +/− 1.8 nM from multiple measurements; see Table 2).

Figure 4

Figure 4

Binding of peptide SB19-C4-FLAG to immobilized streptavidin. [35S]methionine labeled SB-19 was incubated with a range of concentrations of SA for 1 hour before being transferred to an SA-coated plate and incubated for 5 min. The y axis shows the fraction of surface-bound peptide that is competed by the free SA. This experiment was repeated in duplicate and gave a_K_D of 2.4 nM each time.

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