Synthesis and biophysical properties of C5-functionalized LNA (locked nucleic acid)

Abstract

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA's oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3'-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3'-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.

Figure 1

Structure of LNA-T and C5-functionalized analogues thereof studied herein.

Scheme 1. Synthesis of C5-Alkynyl-Functionalized LNA Uridine Phosphoramidites

Abbreviations: CAN, ceric ammonium nitrate; DMTrCl, 4,4′-dimethoxytrityl chloride; PCl, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite; DIPEA, N,N′-diisopropylethylamine.

Scheme 2. Synthesis of C5-Triazolyl-Functionalized LNA Uridine Phosphoramidites

Figure 2

3′-Exonuclease (SVPDE) degradation of singly (left, 3′-CAC BAT ACG) and doubly modified (right, 3′-CAC BAB ACG) C5-functionalized LNA and reference strands. Nuclease degradation studies were performed in magnesium buffer (50 mM Tris-HCl, 10 mM Mg²⁺, pH 9.0) by using 3.3 μM ONs and 0.03 U of SVPDE. Data depicted in the left panel have been previously reported in ref (22).

Figure 3

Steady-state fluorescence emission spectra of single-stranded B5 ONs (5′-CG CAA CBC AAC GC) and the corresponding duplexes with fully complementary or singly mismatched DNA strands (mismatched nucleotide opposite of modification is specified). Conditions: λ_ex 344 nm (V5/Y5/Z5), λ_ex 375 nm (W5), λ_ex 448 nm (X5); T = 5 °C. Note that different axis scales are used.

Figure 4

Fluorescence intensity of single-stranded probes (SSPs) in the presence or absence of complementary or singly mismatched DNA/RNA strands. Mismatched nucleotide opposite of modification is specified. Hybridization-induced increases and discrimination factors (defined as the fluorescence intensity of duplexes with complementary DNA/RNA divided by the intensity of SSPs or duplexes with mismatched DNA/RNA, respectively) are given above the corresponding histograms. Intensity recorded at λ_em 382 nm for Y5/Y5d and λ_em 377 nm for Z5/Z5d at T = 5 °C. Note that different y-axis scales are used.

Figure 5

Fluorescence intensity of single-stranded probes (SSPs) in the presence or absence of complementary or singly mismatched DNA strands. Mismatched nucleotide opposite of modification is specified. Hybridization-induced increases and discrimination factors are given above the corresponding histograms. Target: 5′-CG CAA BZB AAC GC, where B = C/A/G/T for ON5–8, respectively. Intensity recorded at λ_em 377 nm at T = 5 °C.