Identification and Characterization of Second-Generation Invader Locked Nucleic Acids (LNAs) for Mixed-Sequence Recognition of Double-Stranded DNA

Abstract

Development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate and modify genes. Progress has been made with triplex-forming oligonucleotides, PNAs, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome limitations observed with the classic approaches. In 2005, we introduced Invader Locked Nucleic Acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with ‘+1 interstrand zippers’ of 2’-N-(pyren-1-yl)methyl-2’-amino-α-L-LNA monomers. Despite promising preliminary results, progress has been slow due to the synthetic complexity of the building blocks. Here, we describe a study that led to the identification of two simpler classes of Invader monomers. We compare thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2’-amino-α-L-LNA, 2’-N-methyl-2’-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2’-O-(pyren-1-yl)methyl-RNA or 2’-N-methyl-2’-N-(pyren-1-yl)methyl-2’-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA-recognition for applications in molecular biology and nucleic acid diagnostics.

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.…”

“…16 ONs containing a single incorporation in the 5′-G B G ATA TGC context are denoted X1 , Y1 , and Z1 . Similar conventions apply for the B2–B6 series (Table 1).…”

“…In this case, the two pyrene moieties most likely stack inside the duplex core in a similar manner as recently suggested for duplexes modified with 2′- N -(pyren-1-yl)methyl-2′-amino-α-L-LNA monomers; the spatial separation for pyrene-pyrene stacking in the major groove is too large. 15a,16 Dual intercalation would also explain the energetic lability of the B2:B5 duplexes, as a localized region with one intercalator per base pair would ensue, which represents a violation of the ‘nearest neighbor exclusion principle’. 14 …”

“…We therefore decided to evaluate these probes in greater detail using a gel shift assay that we have used to screen other potential Invader monomers with. 13,16 Thus, a digoxigenin (DIG) labeled DNA hairpin (DH) – comprised of a 9-mer double-stranded mixed-sequence stem that is linked by a T 10 loop – was used as a model target (Figure 6a). 33 As expected, the unimolecular nature of DH1 strongly stabilizes the stem region as seen from the significantly higher T m relative to the corresponding linear DNA duplex ( T m = 57.0 °C vs 29.5 °C, respectively; Figure 6b and Table 1).…”

“…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.…”