Biology subjects

Wellington-Oguri, R.

Publications and source records attributed to Wellington-Oguri, R..

6 recordsLinked to original sources

bioRxiv2024

Compact RNA sensors for increasingly complex functions of multiple inputs

Designing single molecules that compute general functions of input molecular partners represents a major unsolved challenge in molecular design. Here, we demonstrate that high-throughput, iterative experimental testing of diverse RNA designs crowdsourced from Eterna yields sensors of increasingly complex functions of input oligonucleotide concentrations. After designing single-input RNA sensors with activation ratios beyond our detection limits, we created logic gates, including challenging XOR and XNOR gates, and sensors that respond to the ratio of two inputs. Finally, we describe the OpenTB challenge, which elicited 85-nucleotide sensors that compute a score for diagnosing active tuberculosis, based on the ratio of products of three gene segments. Building on OpenTB design strategies, we created an algorithm Nucleologic that produces similarly compact sensors for the three-gene score based on RNA and DNA. These results open new avenues for diverse applications of compact, single molecule sensors previously limited by design complexity.

synthetic biology↗

bioRxiv2021

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

synthetic biology↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2020

Theoretical basis for stabilizing messenger RNA through secondary structure design

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. These computational tests were carried out on both model mRNAs and COVID-19 mRNA vaccine candidates. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term superfolder mRNAs. These designs exhibit wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity, and their folding is robust to temperature, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1, and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable. Significance statementMessenger RNA (mRNA) medicines that encode and promote translation of a target protein have shown promising use as vaccines in the current SARS-CoV-2 pandemic as well as infectious diseases due to their speed of design and manufacturing. However, these molecules are intrinsically prone to hydrolysis, leading to poor stability in aqueous buffer and major challenges in distribution. Here, we present a principled biophysical model for predicting RNA degradation, and demonstrate that the stability of any mRNA can be increased at least two-fold over conventional design techniques. Furthermore, the predicted stabilization is robust to post-design modifications. This conceptual framework and accompanying algorithm can be immediately deployed to guide re-design of mRNA vaccines and therapeutics to increase in vitro stability.

biophysics↗

bioRxiv2019

Crowdsourced RNA design discovers diverse, reversible, efficient, self-contained molecular sensors

Internet-based scientific communities promise a means to apply distributed, diverse human intelligence towards previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale videogame-based crowdsourcing of functional RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near-thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs--results that surpass computational and expert-based design. This work represents a new paradigm for widely distributed experimental bioscience. One Sentence SummaryOnline community discovers standalone RNA sensors.

synthetic biology↗

bioRxiv2018

Evidence of an Unusual Poly(A) RNA Signature Detected by High-throughput Chemical Mapping

Homopolymeric adenosine RNA plays numerous roles in both cells and non-cellular genetic material, and for lack of evidence to the contrary, it is generally accepted to form a random coil under physiological conditions. However, chemical mapping data generated by the Eterna Massive Open Laboratory indicates that a poly (A) sequence of length seven or more, at pH 8.0 and MgCl concentrations of 10 mM, develops unexpected protection to selective 2-hydroxyl acylation read out by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing. This protection first appears in poly(A) sequences of length 7 and grows to its maximum strength at length ~10. In a long poly(A) sequence, substitution of a single A by any other nucleotide disrupts the protection, but only for the 6 or so nucleotides on the 5 side of the substitution. The authors are grateful for pre-publication comments; please use https://docs.google.com/document/d/14972Q36IDTYMglwMXTOrqd4P9orQ6-P3bPbCuITdv6A.

biochemistry↗