Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines for Invaders Based on 2′-O-(Pyren-1-yl)methyl-RNA Monomers

Abstract

Development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable due to their potential as tools for detection, regulation and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-O-(pyren-1-yl)methyl-RNA monomers and have been recently shown to recognize linear dsDNA, DNA hairpins and chromosomal DNA. In the present work, we systematically study the role that the nucleobase moieties of the 2′-O-(pyren-1-yl)methyl-RNA monomers have on recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with +1 interstrand zippers of the four canonical 2′-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers, show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprised of C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of effective Invaders for applications in molecular biology, nucleic acid diagnostics and biotechnology.

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

“…In addition, duplexes with +2 interstrand monomer arrangements display prominent and unstructured emission centered at λ em ~490 nm, which is consistent with pyrene excimer emission (Figure 4). 15,16,19,31 The presence of these signals shows that the pyrene moieties are stacking with an interplanar separation of ~3.4 Å. We speculate that stacking occurs in the major groove in a similar manner as previously suggested for duplexes with +2 zipper arrangements of pyrene-functionalized ara -uridine monomers.…”

“…Thus, ONs in which the pyrene-functionalized monomers are flanked by 3′-purines, induce greater stabilization than ONs with 3′-flanking pyrimidines (e.g., compare Δ T m /mod values for B2 - and B4 -series, Table 1). 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.…”

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

“…16,17 Thus, the free nucleosides are first O2′-pyrenylmethylated via Williamson’s approach in modest regioselectivity and yield. This is followed by N -acylation of the exocyclic amines via transient protection protocols, O5′-dimethoxytritylation, and O3′-phosphitylation.…”

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

“…Another characteristic of DNA duplexes with +1 interstrand arrangements of intercalator-modified nucleotide monomers, 13-15 which is shared by S2:S5 , is the blue-shifted pyrene absorption, which is indicative of reduced pyrene-nucleobase interactions due to locally perturbed duplex geometries (compare λ max for S2:S5 with λ max for other probe duplexes, Table 4, or probe-target duplexes, Table 3). Moreover, steady-state fluorescence emission spectra of S2:S5 (and of +2 zipper probe S1:S4 ) exhibit prominent and unstructured emission at λ em ~490 nm, which is consistent with pyrene-pyrene excimers.…”

“…6,13-15 We therefore selected to evaluate the dsDNA-recognition efficiency of S2:S5 using a 3′-digoxigenin (DIG) labeled DNA hairpin (DH) as a model dsDNA target, which is comprised of a 9-mer double-stranded mixed sequence stem linked by a T 10 loop (Figure 4). However, incubation of DH1 with S2:S5 in a HEPES buffer for 12-16 hours at ambient temperatures did not result in formation of slower-migrating recognition complexes on non-denaturing PAGE gels even at 500-fold molar probe excess (Figure 4).…”