Designed Arginine-Rich RNA-Binding Peptides with Picomolar Affinity

Austin, Ryan J.; Xia, Tianbing; Ren, Jinsong; Takahashi, Terry T.; Roberts, Richard W.

doi:10.1021/ja026610b

Cited by 88 publications

(73 citation statements)

References 26 publications

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“…The λ-phage N protein-boxB RNA interaction, which normally regulates antitermination during transcription of λ-phage mRNAs, fits these criteria (17). The λN peptide, which mediates the binding of the N protein, is only 22 amino acids long, and the boxB RNA hairpin that it recognizes is only 17 nucleotides long and they can bind with nanomolar affinity (18). To generate a recombinant editase, the λN peptide was fused to the deaminase domain of human ADAR2 (λN-DD), and recombinant protein was purified from the yeast Pichia pastoris as previously described (19,20).…”

Section: Resultsmentioning

confidence: 95%

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Montiel-González

Vallecillo-Viejo

Yudowski

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

196

198

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Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter protein function. By removing the endogenous targeting domains from human adenosine deaminase that acts on RNA 2 and replacing them with an antisense RNA oligonucleotide, we have engineered a recombinant enzyme that can be directed to edit anywhere along the RNA registry. Here we demonstrate that this enzyme can efficiently and selectively edit a single adenosine. As proof of principle in vitro, we correct a premature termination codon in mRNAs encoding the cystic fibrosis transmembrane conductance regulator anion channel. In Xenopus oocytes, we show that a genetically encoded version of our editase can correct cystic fibrosis transmembrane conductance regulator mRNA, restore fulllength protein, and reestablish functional chloride currents across the plasma membrane. Finally, in a human cell line, we show that a genetically encoded version of our editase and guide RNA can correct a nonfunctional version of enhanced green fluorescent protein, which contains a premature termination codon. This technology should spearhead powerful approaches to correcting a wide variety of genetic mutations and fine-tuning protein function through targeted nucleotide deamination.

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Section: Resultsmentioning

confidence: 95%

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Montiel-González

Vallecillo-Viejo

Yudowski

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

196

198

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“…The N-terminal portion (residues 1-11) anchors the peptide on the RNA target (with M affinity; ref. 29), and the C-terminal portion (residues 12-22) may undergo domain motion between a bend ␣-helix that has Trp-18 stacking at the interface with A7 of RNA and the unstacked complex, where Trp-18 does not stack on A7. One of the unique features of the charge transfer in these RNA-peptide complexes is that the transfer proceeds across the interface between the two biomolecules, RNA, and peptide, and is mediated through a single-stranded stacking structure.…”

Section: Discussionmentioning

confidence: 99%

The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

Xia

Becker

Wan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The N protein from bacteriophage is a key regulator of transcription antitermination. It specifically recognizes a nascent mRNA stem loop termed boxB, enabling RNA polymerase to read through downstream terminators processively. The stacking interaction between Trp-18 of WT N protein and A7 of boxB RNA is crucial for efficient antitermination. Here, we report on the direct probing of the dynamics for this interfacial binding and the correlation of the dynamics with biological functions. Specifically, we examined the influence of structural changes in four peptides on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in different positions), through mutations of critical residues of N peptide (residues 1-22). We then compare their in vivo (Escherichia coli) transcription antitermination activities with the dynamics. The results demonstrate that the RNA-peptide complexes adopt essentially two dynamical conformations with the time scale for interfacial interaction in the two structures being vastly different, 1 ps for the stacked structure and nanosecond for the unstacked one; only the weighted average of the two is detected in NMR by nuclear Overhauser effect experiments. Strikingly, the amplitude of the observed ultrafast dynamics depends on the identity of the amino acid residues that are one helical turn away from Trp-18 in the peptides and is correlated with the level of biological function of their respective full-length proteins.M acromolecular assemblies play critical roles in biological processes, such as expression (1), recognition (2, 3), and catalysis (4, 5). In these assemblies, the interactions are selective in forming unique structures with dynamics governing the functions. One such complex is that of RNA with proteins. It involves the bacteriophage N protein, which binds a cis-acting stem-loop RNA structure (termed boxB) in nascent mRNA coded in the phage genome, and regulates transcription elongation and termination (6-8). With other host protein factors, the complex enables RNA polymerase to read through intrinsic and Rho-dependent terminators in a processive manner (9).NMR structural studies (10-12) demonstrated that the amino terminal of the N protein (N peptide), which features an arginine-rich motif, binds to the boxB RNA hairpin as a bent ␣-helix, and this process enforces the purine-rich boxB RNA pentaloop to adopt a canonical GNRA fold (13, 14) with the fourth purine residue extruded (Fig. 1). One unique interaction featured in this RNA-peptide complex is that of the side chain of the tryptophan residue (Trp-18), which directly stacks on adenine 7, extending the RNA -stack by one residue (Fig. 1). Different peptide ligands can target boxB RNA with strong equilibrium affinities (nanomolar range for dissociation constant) similar to the WT (15), as we have shown by in vitro protein selection via mRNA display (16,17). However, examination of the energetics and structures by NMR, CD, and steady-state fluorescence on some of the selected 14͞15 mutants (Fig. 1) reveal that they have quit...

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“…These mutants were selected as alternative solutions for binding the boxB RNA (Barrick et al 2001), and most of them maintain a high boxB binding affinity that is comparable to that of the wild-type N-peptide (Austin et al 2002). However, these mutations significantly impact the functional capacity of N protein variants.…”

Section: Introductionmentioning

confidence: 99%

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

Zhang

Lee²,

Zhao³

et al. 2010

RNA

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In bacteriophage l, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.

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Designed Arginine-Rich RNA-Binding Peptides with Picomolar Affinity

Cited by 88 publications

References 26 publications

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

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