A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells

Zeng, Fanyi; Peritz, Tiina; Kannanayakal, Theresa; Kilk, Kalle; Eiríksdóttir, Emelía; Langel, Ülo; Eberwine, James

doi:10.1038/nprot.2006.81

Cited by 41 publications

(29 citation statements)

References 17 publications

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“…To generate scFvCD7-9R, scFvCD7Cys (1mg/ml) was mixed with Cys(Npys)-(D-Arg)9 peptide (9R, Anaspec) in 0.1M phosphate buffer (pH 5.5) at a molar ratio of 10 to 1 and gently stirred for 4 h at room temperature (Zeng et al, 2006). Unconjugated 9R was removed by dialysis using a membrane with a MWCO of 10,000.…”

Section: Methodsmentioning

confidence: 99%

T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice

Kumar

Ban

Kim

et al. 2008

Cell

532

427

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SUMMARY Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a novel delivery method in humanized mice, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rγ−/− mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ PBMC. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naïve T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

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Section: Methodsmentioning

confidence: 99%

T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice

Kumar

Ban

Kim

et al. 2008

Cell

532

427

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“…Despite the notable advantages of interactome capture over the above-mentioned purification techniques, this approach is limited to identification of only mRNA-binding proteins. It appears that the PAIR technology has the potential to isolate RBP complexes for any RNAs expressed in living cells (Figure 2C) (13,82,83). This method uses a cell membrane-penetrating peptide to efficiently deliver a linked PNA oligomer, which is complementary to target endogenous RNAs, into living cells.…”

Section: Purification Of In Vitro- and In Vivo-assembled Rna–protein mentioning

confidence: 99%

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

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RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

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“…We incorporated a fluorophore FRET pair into the TIVA-tag to allow visualization of TIVA-tag uptake as well as uncaging in cells. The cytosolic environment cleaves the CPP from the TIVA-tag 17 , trapping the caged TIVA-tag inside the cell. Then, by selective photoactivation of the TIVA-tag in the desired cell or cells using a laser connected to a microscope 19 , the mRNA-capturing moiety is revealed and subsequently anneals to the poly-A tail of cellular mRNA.…”

Section: Resultsmentioning

confidence: 99%

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

et al. 2014

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Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment.

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A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells

Cited by 41 publications

References 17 publications

T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice

T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

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