RESUMO
The chemical cross-linking of complexes of proteins with nucleic acids is often used in structural and mechanistic studies of these oftentimes unstable and transient complexes. To date, no method has been reported for the thiol-based conjugation of proteins with an RNA backbone, mainly because of instability of the modified ribonucleic acid that is functionalized at the phosphodiester and its rapid hydrolysis. Here, we report the site-specific synthesis of stable RNA oligonucleotides with a thiol-bearing linker that was attached to the phosphodiester backbone, where the ribonucleotide at the cross-linking site was either replaced with 2'-deoxy- or 2'-fluororibonucleotide. The utility of this approach was validated in cross-linking tests with RNase H1, a model protein for RNA/DNA binding and key effector in DNA-like antisense drug therapy. Furthermore, scale-up cross-linking and purification of the complexes confirmed that the method is useful for obtaining preparations of protein-RNA/DNA complexes with purity and stability that are suitable for further biochemical and structural studies. The present approach broadens the repertoire of disulfide-based cross-linking strategies and is a novel tool for the stabilization of protein-RNA complexes in which the interaction occurs via the RNA backbone. This methodology may be broadly applicable to studies of otherwise unstable or transient complexes of proteins with RNA and RNA/DNA.
Assuntos
RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Reagentes de Ligações Cruzadas/química , Cistamina/química , Dissulfetos/química , Humanos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , RNA/química , Ribonuclease H/química , Ribonuclease H/genéticaRESUMO
Disulfide conjugation invariably remains a key tool in research on nucleic acids. This versatile and cost-effective method plays a crucial role in structural studies of DNA and RNA as well as their interactions with other macromolecules in a variety of biological systems. In this article we review applications of disulfide-bridged conjugates of oligonucleotides with other (bio)molecules such as peptides, proteins etc. and present key findings obtained with their help.
Assuntos
DNA/química , Dissulfetos/química , Oligonucleotídeos/química , Peptídeos/química , Proteínas/química , RNA/químicaRESUMO
This 2-part article reviews methods of oligonucleotides functionalization with thiol tethers and their consecutive use in conjugation with other (macro)molecules via a disulfide bridge. This relatively inexpensive, robust and reversible method of conjugation of DNAs, RNAs and their analogs holds a prominent position in a modern biochemistry toolbox and therefore there is a wealth of literature on the subject. In part I methods of thiol/disulfide groups introduction into oligonucleotide strands have been systematized and discussed. A digest of conjugation methods is presented as well.