Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Mar 30;96(7):3606-10.
doi: 10.1073/pnas.96.7.3606.

Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

M Blind  1 , W Kolanus, M Famulok
Affiliations

Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

M Blind et al. Proc Natl Acad Sci U S A. .

Abstract

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the beta2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Selection and characterization of CD18cyt-specific RNA aptamers. (A) Construction of the synthetic DNA pool and primary 46-mer amino acid sequence of the complete cytoplasmic domain of β2-integrin (CD18cyt), immobilized to Sepharose and used in the selection. (B) Sequences and predicted secondary structures of individual aptamer clones D20, D28, D31, and D42. Nucleotides shown in bold are part of the randomized region. The boxed aptamer region in clones D28 and D20 were shown in a damage-selection experiment to be minimal requirements for retaining CD18cyt-binding capacity. The sequence D42 was used as a negative control; this aptamer does not show any detectable binding to CD18cyt.
Figure 2
Figure 2
RNA aptamer expression system based on double infection with recombinant vaccinia viruses. (A) Design of the T7-RNA expression cassette. (B) Schematic representation of the vaccinia virus-based cytoplasmatic RNA aptamer expression system. (C) Course of the expression of TR-encoded aptamer in vT7-coinfected Jurkat E6 cells shown by a representative dot-blot analysis for aptamer TR-D31 (Right). Maximum levels of aptamer expression are seen 7 h postinfection. Quantification (Left) was done as follows: total cellular RNA was isolated, transferred to the blotting membrane and hybridized with 5′-32P-labeled oligonucleotide complementary to the 3′-stabilizing stem-loop structure present in all TR constructs. For comparison and quantification, in vitro-transcribed aptamer TR-D20 was treated in the same way. D, Dot blot analysis and quantification of maximally expressed aptamer RNA. Each dot was quantified on a PhosphorImager. Quantification was done as described for C.
Figure 3
Figure 3
Gel mobility-shift assays of endogenous CD18 contained in Jurkat E6 cell lysates bound to its cognate aptamer TR-D20. (A) Gel-shift experiment 1. Lane 1, free TR-D20 aptamer RNA; lane 2, shifted band obtained in presence of Jurkat E6 lysate and 25 μM nonspecific competitor tRNA; lane 3, no shift obtained with negative control sequence TR-D42; lane 4, same as lane 2 with 40 μM nonspecific competitor tRNA; lane 5, same as lane 3 with 40 μM nonspecific competitor tRNA; lane 6, specific supershifted band obtained in the presence of antibody MHM23, which recognizes the β2-extracellular domain of LFA-1; lane 7, specific supershifted band obtained in the presence of antibody MEM170, which recognizes the extracellular domain of the αL-subunit of LFA-1. The band pattern may reflect aptamer/integrin complexes of different stoichiometry. (B) Gel-shift experiment 2 with additional controls. All gel-shift experiments were performed in the presence of 30 μM tRNA as a nonspecific competitor. Lane 1, free TR-D20 aptamer RNA; lane 2, shifted band obtained in presence of Jurkat E6 lysate; lane 3, specific supershifted band obtained in the presence of antibody MHM23; lane 4, no shifted aptamer obtained in the absence of Jurkat E6 cell lysate under conditions identical to those in lane 3; lane 5, specific supershifted band obtained in the presence of antibody MEM170; lane 6, no supershift obtained in the presence of antibody OKT3 directed against the T cell receptor. The experimental conditions are otherwise the same as in lane 2; lane 7, no shifted aptamer obtained in the absence of Jurkat E6 cell lysate under conditions identical to those in lane 6.
Figure 4
Figure 4
Inhibition of PMA-stimulated cell adhesion as a function of aptamer expression. (A) The CD18cyt binder TR-D20 reduces phorbol ester-activated Jurkat cell adhesion to ICAM-1. Jurkat E6 cells infected with recombinant vaccinia viruses were allowed to adhere to plastic dishes coated with a recombinant ICAM-1 chimera as described in Material and Methods. Jurkat E6 cell adhesion to ICAM-1 was superinducible by PMA in the presence of several control viruses (vT7/vTR, wild-type vaccinia virus, vT7, and vTR-D20). However, induction of CD18cyt-specific aptamer expression (vT7/vTR-D20, Right) reduced PMA-stimulated Jurkat E6 cell adhesion but not basal adhesion. These results were independently reproduced at least three times. Percentage reflects values normalized to stimulated vT7/vTR double infection, which was set to 100%. (B) Human PBMC are good targets for protein overexpression by recombinant vaccinia viruses. Cytoplasmic Ig (cIg) fusions of cytohesin-1 subdomains were detected with the help of an FITC-conjugated anti-human Ig antibody preparation in permeabilized PBMC. Upper Left, control infection, no recombinant molecules expressed; Upper Right, cIg-domains alone; Lower Left, cIg-PH; Lower Right, cIg-PH+c-domain. (C) TR-D20 specifically reduces PMA-stimulated adhesion of PBMC to ICAM-1. Aptamers or control proteins were expressed in PBMC by recombinant vaccinia viruses as described above. PMA-stimulated adhesion of these cells to ICAM-1 was equivalent when the TR-D42 aptamer, cIg, or cIg-PH control proteins were expressed, but stimulated cell adhesion was dramatically reduced after expression of the intact PH + c-domain of cytohesin-1 (cIg-PH+c) or of the TR-D20 aptamer.
Figure 5
Figure 5
Mapping of the binding site of aptamers TR-D20, TR-D28, and TR-D31 and the negative control sequence TR-D42 on CD18cyt by using synthetic biotinylated peptide fragments. All gel shifts were carried out in the presence of 25 μM streptavidin to enhance separation of shifted vs. nonshifted RNA. Gel-shift experiments were performed in the presence of 4 nM radiolabeled RNA and 40 μM nonspecific tRNA competitor. Lane 1, free aptamer in the presence of 25 μM streptavidin and 40 μM unspecific competitor tRNA; lanes 2, 4, and 6 or 3, 5, and 7, same as lane 1 in presence of 25 μM or 12.5 μM, respectively, peptides A23 (lanes 2 and 3), B16 (lanes 4 and 5), and C17 (lanes 6 and 7).
See this image and copyright information in PMC

References

    1. Spencer D M, Wandless T J, Schreiber S L, Crabtree G R. Science. 1993;262:1019–1024. - PubMed
    1. Vidal M, Brachmann R K, Fattaey A, Harlow E, Boeke J D. Proc Natl Acad Sci USA. 1996;93:10315–10320. - PMC - PubMed
    1. Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Nature (London) 1996;380:548–550. - PubMed
    1. Huang J, Schreiber S L. Proc Natl Acad Sci USA. 1997;94:13396–13401. - PMC - PubMed
    1. Rondon I J, Marasco W A. Annu Rev Microbiol. 1997;51:257–283. - PubMed

Publication types

LinkOut - more resources