Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design - PubMed (original) (raw)

Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design

Dwaine A Braasch et al. Nucleic Acids Res. 2002.

Abstract

Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10 degrees C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA-DNA chimeras complementary to the 5'-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells.

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Figures

Figure 1

Figure 1

Structure of locked nucleic acid (LNA).

Figure 2

Figure 2

Schematic of luciferase mRNA showing the approximate target sites of LNA and LNA–DNA chimeras used in these studies.

Figure 3

Figure 3

Images of CV-1 cells transfected with a LNA–DNA chimera that was analogous in sequence to XV and labeled with Cy3 fluorophore. All images are magnified 630 times and the field of view is the same. (Left to right) DIC bright field image; staining with the cell permeable nuclear dye Hoechst 32358; localization of Cy3-labeled LNA–DNA chimera; overlay of image of Hoechst 33258 and Cy3-labeled LNA–DNA chimera.

Figure 4

Figure 4

Dose–response curves of antisense gene inhibition by LNAs and LNA–DNA chimeras targeted to (A) the 5′-UTR, (B) the translation start site and (C) the downstream coding region of luciferase mRNA. All points are averages of triplicate determinations and are normalized to an independent measurement of β-galactosidase activity. Capitalized letters within a sequence represent LNA, lower case letters represent DNA bases and underlined bases depict mismatched bases.

Figure 4

Figure 4

Dose–response curves of antisense gene inhibition by LNAs and LNA–DNA chimeras targeted to (A) the 5′-UTR, (B) the translation start site and (C) the downstream coding region of luciferase mRNA. All points are averages of triplicate determinations and are normalized to an independent measurement of β-galactosidase activity. Capitalized letters within a sequence represent LNA, lower case letters represent DNA bases and underlined bases depict mismatched bases.

Figure 4

Figure 4

Dose–response curves of antisense gene inhibition by LNAs and LNA–DNA chimeras targeted to (A) the 5′-UTR, (B) the translation start site and (C) the downstream coding region of luciferase mRNA. All points are averages of triplicate determinations and are normalized to an independent measurement of β-galactosidase activity. Capitalized letters within a sequence represent LNA, lower case letters represent DNA bases and underlined bases depict mismatched bases.

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References

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