Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes

Abstract

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.

FIG. 1.

Structures of LNA, DNA, RNA, PNA, and 2′-O-methyl RNA. The ribose ring of LNA is constrained by a methylene linkage between the 2′ oxygen and the 4′ carbon.

FIG. 2.

Epifluorescence micrographs of DNA and LNA/DNA probe-conferred signals at 0% formamide in hybridization buffer. The same exposure time (250 ms) was used for all probes.

FIG. 3.

Dissociation curves of Eub338 and Eco621 sequences. Formamide (FA) concentration in hybridization buffer was increased instead of elevating hybridization temperature. (A) Eub338 sequence. Eub338, squares; LEub338-1, diamonds; LEub338-2, circles; and LEub338-2v3, triangles. (B) Eco621 sequence. Eco621, squares; LEco621-1, diamonds; LEco621-1d, circles; LEco621-2, inverted triangles; and LEco621-3, triangles.

FIG. 4.

Relationship between FAd and relative intensities of the probes at 0% formamide (FA) used in this study. The number of LNA nucleotides is indicated for each probe. Diamonds, Eub338 sequence; crosses, GAM42a sequence; circles, Eco468 sequence; triangles, Eco621 sequence; inverted triangles, Eco1068 sequence; and squares, Eco227, Eco252, Eco681, and Eco836.