Single‐Round, Multiplexed Antibody Mimetic Design through mRNA Display

Olson, C. Anders; Nie, Jeff; Diep, Jonathan; Al‐Shyoukh, Ibrahim; Takahashi, Terry T.; Al‐Mawsawi, Laith Q.; Bolin, Jennifer M.; Elwell, Angela L.; Swanson, Scott; Stewart, Ron; Thomson, James A.; Soh, H. Tom; Roberts, Richard W.; Sun, Ren

doi:10.1002/ange.201207005

Cited by 13 publications

(13 citation statements)

References 24 publications

(50 reference statements)

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“…the fraction of folded proteins) and function (i.e. binding affinity to IgG-Fc), was measured in a high-throughput manner by coupling mRNA display with Illumina sequencing (see Materials and methods, Figure 1—figure supplement 3 ) ( Roberts and Szostak, 1997 ; Olson et al, 2012 ). The relative frequency of mutant sequences before and after selection allowed us to compute the fitness of each variant relative to the wild type protein (WT).…”

Section: Resultsmentioning

confidence: 99%

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Adaptation in protein fitness landscapes is facilitated by indirect paths

Dai

Olson

et al. 2016

eLife

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The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20 L ) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 20 4 = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve.

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Section: Resultsmentioning

confidence: 99%

“…Affinity selection by mRNA display ( Roberts and Szostak, 1997 ; Olson et al, 2012 ) was performed as described ( Figure 1—figure supplement 3A ) ( Olson et al, 2014 ). Briefly, The DNA library was transcribed by T7 RNA polymerase (Life Technologies) according to manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Adaptation in protein fitness landscapes is facilitated by indirect paths

Dai

Olson

et al. 2016

eLife

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243

258

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“…Accounting for these additional sequences yields an enrichment of 1.1 × 10 3 , assuming 152 DXXDY(A/S)-containing peptides (Eq. (2)), which is on the order of enrichments reported for individual rounds of mRNA (~10 3 ) 39 , phage (~10 3 ) 14 , or cell surface display (~10 4 ) 40,41 .…”

Section: As-ms Recovers High-affinity Ligands From High Dilutionmentioning

confidence: 95%

Ultra-large chemical libraries for the discovery of high-affinity peptide binders

et al. 2020

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High-diversity genetically-encoded combinatorial libraries (10 8 −10 13 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only~10 6 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 10 8 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 10 6-10 8. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.

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“…In this study, relative binding affinity of all single and nearly all double amino acid mutants to IgG-FC was characterized using mRNA display [31]. mRNA display is an in vitro genetic system in which peptides are covalently linked to the encoding mRNAs ( Figure 1A) typically used to evolve novel molecular recognition tools [31,32]. Here, we used deep sequencing combined with mRNA display to monitor the evolution of GB1 mutants in real time after one generation of affinity enrichment ( Figure 1A).…”

Section: High-resolution Gb1 Double-mutant Affinity Profilementioning

confidence: 99%

A Comprehensive Biophysical Description of Pairwise Epistasis throughout an Entire Protein Domain

2014

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SUMMARY Background Non-additivity in fitness effects from two or more mutations, termed epistasis, can result in compensation of deleterious mutations or negation of beneficial mutations. Recent evidence shows the importance of epistasis in individual evolutionary pathways. However, an unresolved question in molecular evolution is how often and how significantly fitness effects change in alternative genetic backgrounds. Results To answer this question we quantified the effects of all single mutations and double mutations between all positions in the IgG-binding domain of protein G (GB1). By observing the first two steps of all possible evolutionary pathways, this fitness profile enabled the characterization of the extent and magnitude of pairwise epistasis throughout an entire protein molecule. Furthermore, we developed a novel approach to quantitatively determine the effects of single mutations on structural stability (ΔΔGU). This enabled determination of the importance of stability effects in functional epistasis. Conclusions Our results illustrate common biophysical mechanisms for occurrences of positive and negative epistasis. Our results show pervasive positive epistasis within a conformationally dynamic network of residues. The stability analysis shows that significant negative epistasis, which is more common than positive epistasis, mostly occurs between combinations of destabilizing mutations. Furthermore, we show that although significant positive epistasis is rare, many deleterious mutations are beneficial in at least one alternative mutational background. The distribution of conditionally beneficial mutations throughout the domain demonstrates that the functional portion of sequence space can be significantly expanded by epistasis.

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Single‐Round, Multiplexed Antibody Mimetic Design through mRNA Display

Cited by 13 publications

References 24 publications

Adaptation in protein fitness landscapes is facilitated by indirect paths

Adaptation in protein fitness landscapes is facilitated by indirect paths

Ultra-large chemical libraries for the discovery of high-affinity peptide binders

A Comprehensive Biophysical Description of Pairwise Epistasis throughout an Entire Protein Domain

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