High-Affinity DNA Targeting Using Readily Accessible Mimics of N2′-Functionalized 2′-Amino-α-L-LNA

Abstract

N2′-Pyrene-functionalized 2′-amino-α-L-LNAs (Locked Nucleic Acids) display extraordinary affinity toward complementary DNA targets due to favorable preorganization of the pyrene moieties for hybridization-induced intercalation. Unfortunately, the synthesis of these monomers is challenging (~20 steps, <3% overall yield), which has precluded full characterization of DNA-targeting applications based on these materials. Access to more readily accessible functional mimics would be highly desirable. Here we describe short synthetic routes toward a series of O2′-intercalator-functionalized uridine and N2′-intercalator-functionalized 2′-N-methyl-2′-aminouridine monomers and demonstrate – via thermal denaturation, UV-visible absorption and fluorescence spectroscopy experiments – that several of them mimic the DNA-hybridization properties of N2′-pyrene-functionalized 2′-amino-α-L-LNAs. For example, oligodeoxyribonucleotides (ONs) modified with 2′-O-(coronen-1-yl)methyluridine monomer Z, 2′-O-(pyren-1-yl)methyluridine monomer Y or 2′-N-(pyren-1-ylmethyl)-2′-N-methylaminouridine monomer Q, display prominent increases in thermal affinity toward complementary DNA relative to reference strands (average ΔTm/mod up to +12 °C), pronounced DNA-selectivity, and higher target specificity than 2′-amino-β-L-LNA benchmark probes. In contrast, ONs modified with 2′-O-(2-napthyl)uridine monomer W, 2′-O-(pyren-1-yl)uridine monomer X or 2′-N-(pyren-1-ylcarbonyl)-2′-N-methylaminouridine monomer S display very low affinity toward DNA targets. This demonstrates that even conservative alterations in linker chemistry, linker length and surface area of the appended intercalators have marked impact on DNA-hybridization characteristics. Straightforward access to high-affinity building blocks such as Q/Y/Z is likely to accelerate their use in DNA-targeting applications within nucleic acid based diagnostics, therapeutics, and material science.

Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

“…17b Thus, treatment of O 2, O 2′-anhydrouridine 1 20 with tris(pyren-2-yl)methyl borate or tris(pyren-4-yl)methyl borate - generated in situ 21 via addition of 2-pyrenemethanol 22 or 4-pyrenemethanol 23 to borane - afforded 2Y and 2Z in modest but acceptable yields (30% and 20% respectively, Scheme 1). Subsequent O5′-dimethoxytritylation gave nucleosides 3Y and 3Z , which upon treatment with 2-cyanoethyl- N , N -diisopropylchlorophosporamidite (PCl reagent) and Hünig’s base provided target phosphoramidites 4Y and 4Z in excellent yield.…”

“…16 The resulting probes display similar dsDNA-recognition efficiency and are much easier to synthesize. 17,18 Identification of these simple building blocks allowed us to initiate systematic structure-property relationship studies with the goal of gaining additional insights into the structural determinants governing Invader recognition efficiency. For example, we demonstrated that all four canonical 2′- O -(pyren-1-yl)methyl-RNA monomers can be used to construct dsDNA-binding Invader probes.…”

25 years and still going strong: 2′-O-(pyren-1-yl)methylribonucleotides – versatile building blocks for applications in molecular biology, diagnostics and materials science

“…Our group found the initial O3′,O5′-tritylation of uridine to be cumbersome and therefore devised an alternative route 12 to key intermediate 3 that is based on trialkylborate 13 mediated ring opening of O2,O2′-anhydrouridine 6 using 1-pyrenemethanol in anhydrous DMSO at high temperatures (Scheme 2). Key intermediate 3 is obtained in slightly higher overall yield (~18% vs ~23%) and this route is, in our opinion, more practical across a wider range of reaction scales.…”

Synthesis and characterization of oligodeoxyribonucleotides modified with 2′-thio-2′-deoxy-2′-S-(pyren-1-yl)methyluridine

“…As it is frequently observed with intercalator-modified ONs, 14,17,26 mismatched DNA targets are less efficiently discriminated than with unmodified reference strands. While S2 and O2 display similar binding fidelity, significantly better discrimination is observed with N2 .…”

“…We hypothesized that the lower electronegativity of the sulfur atom of 2′- S -(pyren-1-yl)methyl-2′-thiouridine monomer S would weaken the gauche effect between O4′ and the 2′-substituent, and thus increase the population of C2′- endo ( South -type) furanose conformations. 16 This, in turn, was expected to result in more favorable conditions for pyrene intercalation, leading to higher cDNA affinity relative to ONs modified with current-generation Invader building blocks 2′- O -(pyren-1-yl)methyluridine monomer O 17 or 2′- N -(pyren-1-yl)methyl-2′- N -methyl-2′-aminouridine monomer N 17 (Figure 1). …”