Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions

Kurz, Markus; Gu, Ke; Lohse, Peter

doi:10.1093/nar/28.18.e83

Cited by 86 publications

(67 citation statements)

References 21 publications

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Section: Methods Mrna Display Library Preparationmentioning

confidence: 99%

“…; where unlabeled bases are 2′-OMe RNA, Ps = psoralen C6, S9 = spacer phosphoramidite 9, and Pu = puromycin-CPG, Glen Research) 26 .…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…The template DNA was transcribed and purified as described previously 25 . Cross-linking of a puromycin-psoralen linker to the mRNA was performed as described using oligo 28A.1 (5′-; where unlabeled bases are 2′-OMe RNA, Ps = psoralen C6, S9 = spacer phosphoramidite 9, and Pu = puromycin-CPG, Glen Research) 26 .In vitro translation with the mRNA-28A.1 library was performed in Red Nova Lysate (Novagen) as per the manufacturer's instructions with optimized conditions (100 mM KOAc, 0.5 mM MgOAc, 0.4 μM mRNA-28A.1, and ~25 μM overall L-methionine; 10 ml total reaction volume) and 35 S-methionine (0.5 mCi ml −1 final). RNA-peptide fusions were salt-treated and purified as described previously 25 .…”

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confidence: 99%

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Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

West

Delker

et al. 2007

Nat Chem Biol

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G protein-coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins 1 . Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains2. Down-regulation of mth increases the lifespan of Drosophila melanogaster 3 -inhibitors of Mth signaling would thus be expected to enhance longevity. We used mRNA display selection 4,5 to identify high affinity (K D = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto-and transmembrane (TM) domains. Flies constitutively expressing a Mth antagonist peptide exhibit a robust lifespan extension, suggesting that the peptides inhibit Mth signaling in vivo. Our work thus provides novel lifespan-extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.Because of their participation in numerous cell processes, GPCRs are the targets of approximately half of marketed drugs, and new GPCR ligands continue to be pursued and developed 6 . Naïve approaches toward GPCR ligand identification typically involve highthroughput screening of a molecular library (10 2 to 10 5 unique members) in functional, cellbased assays [6][7][8] . A powerful, alternative approach for the rapid isolation of novel ligands is in vitro peptide selection using mRNA display, which allows access to very high library complexities (>10 13 ) in a robust format 4 . High affinity peptide ligands for RNA, small molecule, and protein targets have been identified by mRNA display selection 5 .The crystal structure of the hexahistidine-tagged Mth ectodomain was previously determined, demonstrating that the mature, N-terminal extracellular domain of Mth is a stably folded, glycosylated protein of 195 residues 9 . Since the ectodomains of other class B GPCRs maintain recognition of their cognate ligands independently of their TM cores 10,11 , we targeted the Mth ectodomain for in vitro selection to isolate putative modulators of Mth signaling. We expressed and purified a specifically biotinylated construct of the Mth ectodomain 12 to avoid using the weak hexahistidine epitope as an immobilization tag for selection 13 . Hence, the Mth ectodomain was homogeneously presented, perhaps mimicking the juxtaposition of the ectodomain and TM domain in the context of the full-length receptor.We constructed a random, 27-mer peptide mRNA display library. After eight rounds of selection, with the final four rounds including preclearing steps on matrix without target and specific elution with free, non-biotinylated Mth, we obtained a final 8 th round pool that exhibited high activity for Mth and negligible non-specific binding (Fig. 1). DNA sequencing of individual clones from the final selection round revealed a highly conserved consensus, [R/ P]xxWxxR, which we term the RWR motif ...

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Section: Methods Mrna Display Library Preparationmentioning

confidence: 99%

“…; where unlabeled bases are 2′-OMe RNA, Ps = psoralen C6, S9 = spacer phosphoramidite 9, and Pu = puromycin-CPG, Glen Research) 26 .…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

mentioning

confidence: 99%

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Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

West

Delker

et al. 2007

Nat Chem Biol

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“…The resulting double-stranded DNAs were PCR amplified and fractionated. The mRNA-displayed VSMC proteome domain library was generated according to the previously reported procedures (18,19,35). The resulting mRNA-displayed proteome library was successively purified on the basis of the E and FLAG affinity tags at the Nand C termini, respectively, using 10-fold excess of free E and FLAG peptides for competitive elution from the corresponding affinity chromatography resins (termed mRNA display-based preselection).…”

Section: Phosphorylation Of Shps-1/cd Expressed From Insectmentioning

confidence: 99%

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

Shen

Radhakrishnan

et al. 2009

Molecular & Cellular Proteomics

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Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2⅐p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF

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“…The originally described method for RNA-protein fusion preparation involved translation of an enzymatically linked mRNA-puromycin conjugate [3]. Recently, we developed a simplified method for fusion synthesis from psoralen-crosslinked mRNA-puromycin templates [7]. Here we report a further improvement of this method in which the mRNA-puromycin template is prepared in an integrated affinity purification and crosslink formation process on solid phase.…”

Section: Introductionmentioning

confidence: 99%

An Efficient Synthetic Strategy for the Preparation of Nucleic Acid-Encoded Peptide and Protein Libraries for In Vitro Evolution Protocols

Kurz¹,

Kuang²

2000

Molecules

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Abstract:We describe an improved synthetic strategy for the preparation of nucleic acid encoded peptide and protein libraries. A solid-phase format was used to prepare and purify a novel type of mRNA-template for in vitro mRNA-protein fusion synthesis. The present protocol simplifies and accelerates the preparation of fusion libraries and should prove most useful for in vitro protein evolution procedures which involve repetitive cycles of fusion library preparation and selection.

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Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions

Cited by 86 publications

References 21 publications

Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

An Efficient Synthetic Strategy for the Preparation of Nucleic Acid-Encoded Peptide and Protein Libraries for In Vitro Evolution Protocols

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