Random-sequence genetic oligomer pools display an innate potential for ligation and recombination
- MRC Laboratory of Molecular Biology, United Kingdom
- Katholieke Universiteit Leuven, Belgium
Abstract
Recombination, the exchange of information between different genetic polymer strands, is of fundamental importance in biology for genome maintenance and genetic diversification mediated by dedicated recombinase enzymes. Here, we describe a pervasive non-enzymatic capacity for recombination (and ligation) in random-sequence genetic oligomer pools. Specifically, we examine random and semi-random eicosamer (N20) pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids). While DNA, ANA and HNA pools proved inert, RNA and AtNA pools displayed diverse modes of spontaneous intermolecular recombination, connecting recombination mechanistically to the vicinal ring cis-diol configuration shared by RNA and AtNA. Thus, the chemical constitution that renders both susceptible to hydrolysis emerges as the fundamental determinant of an innate capacity for recombination, which is shown to enable a concomitant increase in compositional, informational and structural pool complexity and hence evolutionary potential.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for: Figure 1, Figure 1-supplement 1, Figure 2, Figure 2-supplement 1, Figure 4, Figure 4-supplement 2, Figure 4-supplement 3, Figure 5, Figure 5-supplement 1, Figure 6 and Figure 6-supplement 2. Further source files are shell scripts for the motif search, extraction of secondary structure frequencies and size distributions and python scripts for the generation of random sequences using position-specific nucleotide frequencies and the simulations of the population level Shannon Index at different cleavage / ligation rates are available online (see Source Code Files 1-5).
-
Data from: Random-sequence genetic oligomer pools display an innate potential for ligation and recombinationDryad Digital Repository, 10.5061/dryad.gj8jv50.
Article and author information
Author details
Alexander I Taylor
Funding
Medical Research Council (MC_U105178804)
- Alexander I Taylor
- Benjamin T Porebski
- Gillian Houlihan
- Philipp Holliger
Federation of European Biochemical Societies (FEBS Long-Term Fellowship)
- Hannes Mutschler
KU Leuven (OT/1414/128)
- Mikhail Abramov
- Piet Herdewijn
FWO Vlaanderen (G078014N)
- Mikhail Abramov
- Piet Herdewijn
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Ulrich Muller, UCSD, United States
Version history
- Received: October 21, 2018
- Accepted: November 16, 2018
- Accepted Manuscript published: November 21, 2018 (version 1)
- Version of Record published: December 11, 2018 (version 2)
Copyright
© 2018, Mutschler et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,366
- views
-
- 471
- downloads
-
- 42
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Random-sequence genetic oligomer pools display an innate potential for ligation and recombination
eLife 7:e43022.
https://doi.org/10.7554/eLife.43022
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.
-
- Biochemistry and Chemical Biology
Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
