High-Resolution Molecular Discrimination by RNA

Jenison, Robert; Gill, Stanley C.; Pardi, Arthur; Polisky, Barry

doi:10.1126/science.7510417

Cited by 1,096 publications

(945 citation statements)

References 23 publications

Supporting

Mentioning

908

Contrasting

Unclassified

Order By: Relevance

“…We made K d measurements by equilibrium dialysis or spin-filtration 14 , with equivalent results, using purified MBP fusion proteins exchanged into selection binding buffer by gel filtration. We determined the K d for ATP and ATP analogues by displacing bound 32 P-ATP from the protein by increasing concentrations of competitor.…”

Section: K D Determinationsmentioning

confidence: 99%

See 1 more Smart Citation

Functional proteins from a random-sequence library

Keefe

Szostak

2001

Nature

501

364

View full text Add to dashboard Cite

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...

show abstract

Section: K D Determinationsmentioning

confidence: 99%

“…The inputs into rounds 10-12 were subjected to mutagenic PCR amplification with an average mutagenesis rate of 3.7% at the amino-acid level per amplification. Dissociation constants of protein-ATP (and ATP analogue) complexes as determined by displacement equilibrium filtration 14 for protein 18-19 as an MBP fusion protein.…”

Section: Supplementary Materialsmentioning

confidence: 99%

Functional proteins from a random-sequence library

Keefe

Szostak

2001

Nature

501

364

View full text Add to dashboard Cite

show abstract

“…In the first approach, an in vitro selected theophylline-binding aptamer (see ref. 11 ) was fused via a short spacer region to a sequence complementary to the 3 0 -part of the aptamer. Together with this aptamer part, the spacer region and the complementary sequence lead to the formation of a stable intrinsic terminator in the absence of the aptamer ligand.…”

Section: Introductionmentioning

confidence: 99%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

View full text Add to dashboard Cite

Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

show abstract

“…5A). 44 In TBSV a theophylline aptamer-based attenuation structure was used to replace the native attenuation structure for sg mRNA2 (Fig. 5B).…”

Section: Perspectivesmentioning

confidence: 99%