UNA (unlocked nucleic acid): A flexible RNA mimic that allows engineering of nucleic acid duplex stability

https://doi.org/10.1016/j.bmc.2009.06.045Get rights and content

Abstract

UNA (unlocked nucleic acid) monomers are acyclic derivatives of RNA lacking the C2′–C3′-bond of the ribose ring of RNA. Synthesis of phosphoramidite UNA building blocks of the nucleobases adenine, cytosine, guanine, and uracil is described herein together with their incorporation into RNA strands. UNA monomers additively decrease nucleic acid duplex stability and can be positioned strategically to induce either lack of discrimination of mismatches, that is, universal base behavior, or increased discrimination of mismatches, that is, improved hybridization specificity. UNA-modified RNA duplexes are shown to structurally mimic unmodified RNA duplexes by CD spectroscopy.

Graphical abstract

UNA monomers (‘unlocked RNA’) enable modulation of nucleic acid duplex stability and base pairing specificity, and have properties that are antipodal to those of LNA monomers (‘locked RNA’).

  1. Download : Download full-size image

Introduction

Chemical modification of the natural nucleotide monomers has been widely explored to engineer nucleic acid binding affinity and biological activity of synthetic oligonucleotides (ONs).1, 2, 3, 4 LNA (locked nucleic acid)5, 6, 7, 8, 9 monomers (Fig. 1) are well known for their affinity increasing effect, and short LNA-containing probes for their excellent binding specificity, that is, capability to discriminate effectively between fully matched and singly mismatched targets.10 LNA-modified duplexes formed with RNA complementary strands are of the A-type and structurally very similar to the corresponding unmodified RNA duplexes as has been shown by CD and NMR spectroscopy.11, 12

UNA (unlocked nucleic acid) is an acyclic analogue of RNA in which the bond between the C2′ and C3′ atoms of the ribose ring has been cleaved (Fig. 1). In 1995 we introduced the thymine UNA monomer as a modification in DNA oligonucleotides together with a method for synthesis of the corresponding phosphoramidite derivative.13 Since then, UNA monomers have been incorporated into DNA-type oligonucleotides using O2′-silylated phosphoramidites.14 Furthermore UNA-adenylate trimers were synthesized and shown to be stable against phosphordiesterases.15, 16 It should be noted that seco-RNA has been used as a term to describe various acyclic forms of RNA and 2′,3′-seco-RNA as a term to describe what herein is termed as UNA.13, 14 Along with many other acyclic nucleotide modifications, UNA was shown to induce decreased binding affinity towards a complementary DNA strand,13 but UNA-modified RNA strands or duplexes were not described. UNA has been explored as a constituent in the central DNA segment of gap-mer antisense oligonucleotides, and compatibility with RNase H recognition and RNA cleavage was reported.14

Both UNA and LNA can be described as RNA analogues. However, whereas the additional methylene unit linking the O2′ and C4′ atoms of LNA monomers locks the furanose ring into a C3′-endo conformation, the acyclic nature of UNA renders this molecule very flexible. Therefore LNA and UNA can be considered ‘locked RNA’ and ‘unlocked RNA’, respectively. These antipodal structural characteristics make UNA and LNA complementary with respect to effect on binding affinity towards DNA targets.5, 6, 7, 13

We have initiated a program to broadly evaluate the influence of UNA monomers on hybridization thermodynamics and nucleic acid duplex structures, and to explore their use within biotechnology. Herein we introduce the synthesis of O2′-benzoylated UNA phosphoramidite derivatives of all four standard RNA nucleobases and demonstrate their full compatibility with automated RNA synthesis. We reveal that UNA-modified RNA:RNA duplexes retain A-type structures and that strategic UNA modification of 21-mer RNA strands allow predictable decrease of RNA:RNA or RNA:DNA duplex stabilities and wide-spectrum modulation of hybridization specificity.

Section snippets

Results and discussion

UNA phosphoramidites were synthesized by an optimized version of the published procedure13 for synthesis of the thymine monomer. O5′-DMT protected ribonucleosides 1 were treated with sodium periodate to induce oxidative diol cleavage of the bond linking the C2′ and C3′ atoms of the ribose ring. Subsequent reduction by sodium borohydride yielded the acyclic nucleosides 2. Selective benzoylation of O2′ was optimized relative to the previously published procedure for the thymine derivative by

Conclusion

Synthesis of UNA phosphoramidite derivatives suitable for highly efficient incorporation of UNA monomers of the four natural bases into RNA strands has been accomplished. UNA monomers destabilize duplexes formed between UNA-modified RNA strands and complementary RNA or DNA, and two UNA monomers can be used to decrease or increase mismatch discrimination depending on their sites of incorporation. UNA has herein been introduced as an RNA mimicking novel tool for engineering of nucleic acid duplex

General

Reactions under anhydrous conditions were carried out under an atmosphere of nitrogen. After column chromatographic purifications, the fractions containing product were pooled and evaporated to dryness under reduced pressure. After drying organic phases over Na2SO4, filtration was performed. Solvents were of HPLC grade, of which acetonitrile, DCM and pyridine were dried over molecular sieves (4 Å). TLC was run on Merck Silica 60 F254 aluminum sheets. 1H NMR spectra were recorded at 300 MHz, 13C

Acknowledgments

The Nucleic Acid Center is a research center of excellence funded by the Danish National Research Foundation for studies on nucleic acid chemical biology. We gratefully acknowledge financial support by The Danish National Research Foundation and the Sixth Research Framework Programme of the European Union, Project RIGHT (LSHB-CT-2004 005276).

References and notes (29)

  • A.A. Koshkin et al.

    Tetrahedron

    (1998)
  • P. Nielsen et al.

    Bioorg. Med. Chem.

    (1995)
  • A.V. Itkes et al.

    FEBS Lett.

    (1988)
  • S.N. Mikhailov et al.

    Tetrahedron Lett.

    (1985)
  • S.M. Freier et al.

    Nucleic Acids Res.

    (1997)
  • J. Wengel

    Acc. Chem. Res.

    (1999)
  • J. Micklefield

    Curr. Med. Chem.

    (2001)
  • J. Kurreck

    Eur. J. Biochem.

    (2003)
  • S. Obika et al.

    Tetrahedron Lett.

    (1998)
  • B. Vester et al.

    Biochemistry

    (2004)
  • J.S. Jepsen et al.

    Curr. Opin. Drug Discovery

    (2004)
  • H. Ørum et al.

    Curr. Opin. Mol. Ther.

    (2001)
  • H. Ørum et al.

    Clin. Chem.

    (1999)
  • M. Petersen et al.

    J. Am. Chem. Soc.

    (2002)
  • Cited by (102)

    • Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs

      2022, Molecular Therapy Nucleic Acids
      Citation Excerpt :

      Whereas 8-bromoguanosine derivatives introduced in G12 slightly influenced the ribozyme activity, RH1-12-4 showed a reaction rate similar to that of RH1-12-7, with a kobs equal to 0.002 min−1 (Table 4). Moreover, modification in hairpin loop L2 with unlocked nucleic acid (UNA) analogs of uridine and isoguanosine46–48 resulted in an almost unchanged reaction rate for RH1-L10 but almost completely stopped the reaction for RH1-L11. This result indicates that the isoguanosine residue in the loop significantly changed the interaction in the ribozyme, which led to nearly no cleavage activity by ribozyme RH1-L11 (Table 4).

    View all citing articles on Scopus
    View full text