Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these properties, aptamers have been slow to reach the marketplace, with only one aptamer-based drug receiving approval so far. A series of aptamers currently in development may change how nucleic acid therapeutics are perceived. It is likely that in the future, aptamers will increasingly find use in concert with other therapeutic molecules and modalities.
DNA-encoded chemical library technologies are increasingly being adopted in drug discovery for hit and lead generation. DNA-encoded chemistry enables the exploration of chemical spaces four to five orders of magnitude more deeply than is achievable by traditional high-throughput screening methods. Operation of this technology requires developing a range of capabilities including aqueous synthetic chemistry, building block acquisition, oligonucleotide conjugation, large-scale molecular biological transformations, selection methodologies, PCR, sequencing, sequence data analysis and the analysis of large chemistry spaces. This Review provides an overview of the development and applications of DNA-encoded chemistry, highlighting the challenges and future directions for the use of this technology.
Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3′ end of its encoding mRNA 1 , to sample a large number of distinct random sequences. Starting from a library of 6 × 10 12 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries 2,3 .The frequency of occurrence of functional proteins in collections of random sequences is an important constraint on models of the evolution of biological proteins. Here we have experimentally determined this frequency by isolating proteins with a specific function from a large random-sequence library of known size. We selected for proteins that could bind a small molecule target with high affinity and specificity as a way of identifying amino-acid sequences that could form a three-dimensional folded state with a well-defined binding site and therefore exhibit an arbitrary specific function. ATP was chosen as the target for binding to allow comparison with known biological ATP-binding motifs and also with previous selections using random-sequence RNA libraries 2,3 .Because protein sequences with specific functions are expected to be quite rare in protein sequence space, we prepared a DNA library of 4 × 10 14 independently generated random sequences. This DNA library was specifically constructed to avoid stop codons and frameshift mutations 4 , and was designed for use in mRNA display 1 selections. This DNA library was then used to generate 6 × 10 12 purified non-redundant random proteins that were used as the input into the first selection step. These proteins contain a contiguous stretch of random amino acids 80 residues in length, long enough to form known protein domains. © 2001 Macmillan Magazines LtdCorrespondence and requests for materials should be addressed to J.W.S. (szostak@molbio.mgh.harvard.edu). Supplementary information is available on Nature's World-Wide Web site (http://www.nature.com) or as paper copy from the London editorial office of Nature.The DNA sequences encoding the consensus protein sequences of families A, B, C, D, 18predom and clone 18-19 have been deposited in GenBank under accession codes AF306524 to AF306529, respectively. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptUnlike other libraries that have been used in protein selections, this random region is not part of a larger structure that would otherwise tend to constrain or bias the conformation of th...
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 Ϸ10 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 antibodyantigen 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.affinity tag ͉ peptide library ͉ peptide aptamer ͉ Strep-tag ͉ streptavidin
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.