Recombinant RNA technology: the tRNA scaffold.

Cristallographie & RMN Biologiques, Université Paris Descartes, CNRS, 4 avenue de l'Observatoire, 75006, Paris, France.
Nature Methods (Impact Factor: 23.57). 08/2007; 4(7):571-6. DOI: 10.1038/nmeth1058
Source: PubMed

ABSTRACT RNA has emerged as a major player in most cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA have been in vitro syntheses. Here we describe a generic approach for expressing and purifying structured RNA in Escherichia coli, using tools that parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the 'tRNA scaffold', in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular RNases. This opens the way to large-scale structural and molecular investigations of RNA function.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Many RNAs present unique challenges in obtaining material suitable for structural or biophysical characterization. These issues include synthesis of chemically and conformationally homogeneous RNAs, refolding RNA purified using denaturing preparation techniques, and avoiding chemical damage. To address these challenges, new methodologies in RNA expression and purification have been developed seeking to emulate those commonly used for proteins. In this review, recent developments in the preparation of high-quality RNA for structural biology and biophysical applications are discussed, with an emphasis on native methods.
    Current Opinion in Structural Biology 02/2014; 26C:1-8. · 8.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Modulation of immune response is important in cancer immunotherapy, vaccine adjuvant development and inflammatory or immune disease therapy. Here we report the development of new immunomodulators via control of shape transition among RNA triangle, square and pentagon. Changing one RNA strand in polygons automatically induced the stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares and pentagons. When immunological adjuvants were incorporated, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced in vitro and in animals up to 100-fold, while RNA polygon controls induced unnoticeable effect. The RNA nanoparticles were delivered to macrophages specifically. The degree of immunostimulation greatly depended on the size, shape and number of the payload per nanoparticles. Stronger immune response was observed when the number of adjuvants per polygon was increased, demonstrating the advantage of shape transition from triangle to pentagon.
    Nucleic Acids Research 08/2014; · 8.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Crystallization of RNAs with complex three-dimensional architectures remains a formidable experimental challenge. We review a number of successful heuristics involving engineering of the target RNAs to facilitate crystal contact formation, such as those that enabled the crystallization and structure determination of the cognate tRNA complexes of RNase P holoenzyme and the Stem I domain of the T-box riboswitch. Recently, RNA-targeted antibody Fab fragments and Kink-turn binding proteins have joined the ranks of successful chaperones for RNA crystallization. Lastly, we review the use of structured RNAs to facilitate crystallization of RNA-binding proteins and other RNAs.
    Current Opinion in Structural Biology 03/2014; 26C:9-15. · 8.74 Impact Factor

Full-text (2 Sources)

Available from
May 16, 2014