Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-l-LNA Adenine Monomers: High-Affinity Targeting of Single-Stranded DNA

Abstract

Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications.

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

“…This is indicative of 3′-intercalative binding modes of the pyrene moieties, 19,24 as 3′-flanking purines would provide larger π–stacking surfaces than 3′-pyrimidines. Less stable duplexes are formed with cRNA (Δ T m /mod = −8.0 to +7.5 °C, Table 1; trend: Y > X > Z ), which also points to intercalative pyrene binding modes, 18,24a,25 as intercalators generally favor the less compressed B -type helix geometry of DNA:DNA duplexes. 26 …”

“…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.…”

Aging and SKN-1-dependent Loss of 20S Proteasome Adaptation to Oxidative Stress inC. elegans

“…The following chromosomal arrays were also used: P.pbs-5::GFP and P.pbs-5(ARE)::GFP. Due to recent evidence that the DNA synthesis inhibitor fluorodeoxyuridine, commonly used to block egg laying in aging experiments, induces adaptation and promotes cytoprotection in response to stress (15)(16)(17), we chose to transfer adults to new plates on a daily basis to avoid confounding the current generation with its progeny. We also chose to avoid the use of sterile mutants as studies have found that blocking germ line development promotes protein homeostasis (18).…”

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

“…Wengel, Hrdlicka, and coauthors reported N 2′-pyrene-modified 2′-amino-α-L-LNA monomers (T [ 241 , 242 ] and A [ 243 ]) (monomers L , Figure 37 ).…”

“…The first promising results have been described for Invader LNA probes, i.e., a double-stranded Invader probe modified with 2′- N -(pyren-1-yl)methyl-2′-amino-α- l -LNA thymine monomer (monomer L1 , Figure 37 ) [ 17 , 237 ]. The 2′- N -(pyren-1-yl)methyl-2′-amino-α- l -LNA monomers, being incorporated into different positions of oligonucleotides, strongly improve the binding affinity of the probes toward DNA targets (Δ T m /modification was up to +19 °C for T [ 241 , 242 ] and +14 °C for A [ 243 ]). The binding of the probes to complementary targets can be monitored by the significant loss of fluorescence signal, that together with excellent thermal stabilization confirms intercalating binding mode of pyrene moiety in the DNA:DNA duplexes.…”