Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

Kai Hoehlig,Sven Klussmann,Lucas Bethge,John J Rossi

doi:10.1371/journal.pone.0115328

Kai Hoehlig, Sven Klussmann + Show 2 more

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https://doi.org/10.1371/journal.pone.0115328

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Journal: PloS one	Publication Date: Feb 13, 2015
Citations: 47	License type: CC BY 4.0

Affiliation: Noxxon Pharma (Germany)

Abstract

A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

Highlights

Stereospecificity of chemical and enzymatic reactions is a basic principle of thechemistry in our homochiral world [1]
D-amino acids which are found in prokaryotic cell walls and in peptides from higher organisms exclusively derive from post-translational modifications or ribosome-independent peptide synthesis [2]
To explore whether this premise would still be true for heterochiral RNA/ribozyme or RNA/DNAzyme interactions we used enantiomeric oligonucleotides complementary to the 14 nt RNA target sequence and firstly measured temperature-dependent hyperchromicity

Summary

Introduction

Stereospecificity of chemical and enzymatic reactions is a basic principle of the (bio)chemistry in our homochiral world [1]. The translational apparatus for ribosomal protein synthesis is a stereospecific process that only allows for the incorporation of L-amino acids into proteins. D-amino acids which are found in prokaryotic cell walls and in peptides from higher organisms exclusively derive from post-translational modifications or ribosome-independent peptide synthesis [2]. Nucleic acids occur only in their D-configuration in nature.

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