in vitro evolution
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Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 32
Author(s):  
Kohtoh Yukawa ◽  
Ryo Mizuuchi ◽  
Norikazu Ichihashi

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.


2021 ◽  
Author(s):  
Oran Melanker ◽  
Pierre A Goloubinoff ◽  
Gideon Schreiber

Evolution is driven by random mutations, whose fitness outcome is tested over time. In vitro evolution of a library of a randomly mutated protein mimics this process, however, on a short time scale, driven by a specific outcome (such as binding to a bait). Here, we used directed in vitro evolution to investigate the role of molecular chaperones in curbing promiscuity in favor of specificity of protein-protein interactions. Using yeast surface display, we generated a random library of the E. coli protein Uracil glycosylase (UNG), and selected it against various baits. Those included the purified chaperones GroEL, DnaK+DnaJ+ATP, or total protein extracts from WT or delta DnaK+DnaJ cells. We show that in-vitro evolution differs from natural evolution in cells, both physically and thermodynamically. We found that chaperones, whether purified or as part of the protein extract, select for and thus enrich uracil glycosylase (UNG) misfolded species during this in vitro evolution process. In a more general context, our results show that chaperones purge promiscuous misfolded clones from the system, and thereby avoiding their detrimental effects, such as forming wrong interactions with other macromolecules, including proteins, which can harm proteostasis.


Author(s):  
Christi L. McElheny ◽  
Erin L. Fowler ◽  
Alina Iovleva ◽  
Ryan K. Shields ◽  
Yohei Doi

Cefiderocol, a newly approved cephalosporin agent with an extensive spectrum of activity against Gram-negative bacteria, binds siderophore and uses its receptors to access the bacterial periplasm. Loss of functional CirA, an iron transporter, has been associated with cefiderocol resistance.


Author(s):  
Jiří Zahradník ◽  
Shir Marciano ◽  
Maya Shemesh ◽  
Eyal Zoler ◽  
Daniel Harari ◽  
...  

2021 ◽  
Vol 68 ◽  
pp. 129-134
Author(s):  
Cher Ling Tong ◽  
Kun-Hwa Lee ◽  
Burckhard Seelig

2021 ◽  
Author(s):  
Gideon Schreiber ◽  
Jiri Zahradník ◽  
Shir Marciano ◽  
Maya Shemesh ◽  
Eyal Zoler ◽  
...  

Abstract SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the “European” (20E-EU1), “British” (501.V1),”South African” (501.V2), and Brazilian variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9Å Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.


2021 ◽  
Author(s):  
Antonietta Lillo ◽  
Geoffrey Waldo ◽  
Nileena Velappan ◽  
Hau Nguyen
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