DNA strands with alternating incorporations of LNA and 2'- O-(pyren-1-yl)methyluridine: SNP-discriminating RNA detection probes

Abstract

Detection of nucleic acids using fluorophore-modified oligonucleotides forms the basis of many important applications in molecular biology, genetics and medical diagnostics. Here we demonstrate that DNA strands with central segments of alternating locked nucleic acid (LNA) and 2'-O-(pyren-1-yl)methyluridine monomers display very large and highly mismatch-sensitive increases in fluorescence emission upon RNA hybridization, whereas corresponding "LNA-free" controls do not. Absorbance spectra strongly suggest that LNA-induced conformational tuning of flanking 2'-O-(pyren-1-yl)methyluridine monomers places the reporter group in the minor groove upon RNA binding, whereby pyrene-nucleobase interactions leading to quenching of fluorescence are minimized. Accordingly, these easy-to-synthesize probes are promising SNP-discriminating RNA detection probes.

Figure 1

Structures of 2'-O-(pyren-1-yl)methyluridine, conventional LNA, and Glowing LNA monomers.

Figure 2

Steady-state fluorescence emission spectra of ON1–ON3 and the corresponding duplexes with DNA/RNA targets. Spectra were recorded at T = 5 °C using λ_ex = 350 nm and each strand at 1.0 µM concentration in T_m buffer.

Figure 3

Fluorescence intensity of ON3–ON6 and the corresponding duplexes with DNA or RNA targets as measured at λ_em = 376 nm. Hybridization-induced increases/decreases – defined as the intensity ratio between a duplex and single-stranded probe (SSP) – are listed above corresponding histograms. Spectra were recorded at T = 5 °C using λ_ex = 350 nm and each strand at 1.0 µM concentration in T_m buffer. For spectra, see Figs. 2 & S2^†.

Figure 4

Fluorescence intensity of single-stranded probes and duplexes with complementary or centrally mismatched RNA targets as measured at λ_em = 376 nm (mismatched nucleotide listed in parenthesis). Hybridization-induced increases (defined as the intensity ratio between a matched duplex and single-stranded probe) and discrimination factors (defined as the intensity ratio between a matched and mismatched duplex) are listed above the corresponding histograms. Spectra were recorded at T = 5 °C using λ_ex = 350 nm and each strand at 1.0 µM concentration in T_m buffer. For spectra, see Fig S6^†.

Figure 5

Principle of SNP-discriminating RNA detection probes reported herein. Upper: Y-modified ‘LNA-free’ reference probes do not result in substantial fluorescence changes upon hybridization with RNA targets due to intercalation of pyrene. Lower: DNA strands with alternating incorporations of LNA and Y monomers result in large hybridization-induced increases in emission with complementary RNA (pyrene in minor groove) but not with mismatched targets (intercalation of pyrene). Droplets represent pyrene moiety of monomer Y. ‘L’, ‘cRNA’ and ‘mmRNA’ denote conventional LNA monomer, complementary RNA, and centrally mismatched RNA target, respectively.