PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high‐throughput sequencing

Reyes, Sabrina Galiñanes; Kuruma, Yutetsu; Fujimi, Mai; Yamazaki, Masako; Eto, Sumie; Nishikawa, Shota; Tamaki, Satoshi; Kobayashi, Asaki; Mizuuchi, Ryo; Rothschild, Lynn J.; Ditzler, Mark A.; Fujishima, Kosuke

doi:10.1002/bit.27696

Cited by 10 publications

(7 citation statements)

References 68 publications

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“…The oligonucleotide libraries were incorporated into a genotype-phenotype linked construct for mRNA display using a previously published protocol (Fig. S1B) (Reyes et al 2021). Upon transcription and translation of the constructs, the best binding variants were selected against an immobilized 58rRNA, performing 10 and 16 rounds of the selections for libraries M and E, respectively.…”

Section: Resultsmentioning

confidence: 99%

In vitro evolution reveals primordial RNA-protein interaction mediated by metal cations

Giacobelli

Fujishima

Lepšı́k

et al. 2021

Preprint

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RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal L11 C-terminal domain was selected from a ~10^10 library of partially randomized sequences, all composed of 10 prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with MD simulations uncover qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant L11 domain uses bridging ion (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues was compensated by acidic residues plus metal ions.

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Section: Resultsmentioning

confidence: 99%

In vitro evolution reveals primordial RNA-protein interaction mediated by metal cations

Giacobelli

Fujishima

Lepšı́k

et al. 2021

Preprint

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“…We followed the basic protocols of the PURE mRNA display method and made minor updates for this study . For details, see the Supporting Methods.…”

Section: Methodsmentioning

confidence: 99%

“…We followed the basic protocols of the PURE mRNA display method and made minor updates for this study. 38 For details, see the Supporting Methods . In brief, RNA libraries were produced by in vitro transcription from a DNA library, followed by puromycin-DNA-tag ligation.…”

Section: Methodsmentioning

confidence: 99%

“…mRNA display is a widely used in vitro selection technique to rapidly isolate functional peptides from libraries of more than 10 13 molecules. It has been applied to directed evolution studies, drug development, and cellular proteomics analysis. , mRNA display uses a stable covalent link between mRNA and its cognate polypeptide, thus enabling a selection for versatile protein functions such as protein binding, ATP-binding, and catalytic function including RNA ligation. − mRNA display platform has also been diversified, for example, using a cell-free system to analyze protein–protein interaction and screening enzyme inhibitors. , More recently, mRNA display combined with a reconstituted cell-free translation system (PURE mRNA display) has achieved high efficiency in in vitro selection by minimizing the carry-over of intrinsic cellular components. ,, However, there is an apparent downside for the mRNA display when it comes to RNA binding. Especially if the target RNA contains a stretch of a single-stranded region (>5 nt), it can form a stable base-pairing between the mRNA, and therefore, mRNA harboring complementary sequences of the targeted RNA will be selected and hinder the selection process.…”

Section: Introductionmentioning

confidence: 99%

“… 35 , 36 More recently, mRNA display combined with a reconstituted cell-free translation system (PURE mRNA display) has achieved high efficiency in in vitro selection by minimizing the carry-over of intrinsic cellular components. 29 , 37 , 38 However, there is an apparent downside for the mRNA display when it comes to RNA binding. Especially if the target RNA contains a stretch of a single-stranded region (>5 nt), it can form a stable base-pairing between the mRNA, and therefore, mRNA harboring complementary sequences of the targeted RNA will be selected and hinder the selection process.…”

Section: Introductionmentioning

confidence: 99%

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De Novo Single-Stranded RNA-Binding Peptides Discovered by Codon-Restricted mRNA Display

Nishikawa,

Watanabe,

Terasaka

et al. 2023

Biomacromolecules

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RNA-binding proteins participate in diverse cellular processes, including DNA repair, post-transcriptional modification, and cancer progression through their interactions with RNAs, making them attractive for biotechnological applications. While nature provides an array of naturally occurring RNA-binding proteins, developing de novo RNA-binding peptides remains challenging. In particular, tailoring peptides to target single-stranded RNA with low complexity is difficult due to the inherent structural flexibility of RNA molecules. Here, we developed a codon-restricted mRNA display and identified multiple de novo peptides from a peptide library that bind to poly(C) and poly(A) RNA with K D s ranging from micromolar to submicromolar concentrations. One of the newly identified peptides is capable of binding to the cytosine-rich sequences of the oncogenic Cdk6 3′UTR RNA and MYU lncRNA, with affinity comparable to that of the endogenous binding protein. Hence, we present a novel platform for discovering de novo single-stranded RNA-binding peptides that offer promising avenues for regulating RNA functions.

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Sequencing the origins of life

2022

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PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high‐throughput sequencing

Cited by 10 publications

References 68 publications

In vitro evolution reveals primordial RNA-protein interaction mediated by metal cations

In vitro evolution reveals primordial RNA-protein interaction mediated by metal cations

De Novo Single-Stranded RNA-Binding Peptides Discovered by Codon-Restricted mRNA Display

Sequencing the origins of life

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