Abstract
Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′-aminouridine and 2′-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms.
Highlights
The promise of molecular tools that enable detection, regulation, and manipulation of genes, continues to inspire nucleic acid chemists to develop probes for sequence-unrestricted recognition of double-stranded DNA [1,2,3,4,5]
We have recently presented an alternative approach, which is based on short DNA duplexes that are energetically activated for mixed-sequence recognition of double-stranded DNA (dsDNA) through installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′aminouridine monomer X or 2′-O-(pyren-1-yl)methyluridine monomer Y (Figure 1—for a definition of the zipper nomenclature, see the Experimental Section) [18,19]
In order to delineate the role of backbone chemistry, X- and Y-modified PS-DNA were studied in the same 9- and 13-mer nucleotide sequence contexts that were used to study the corresponding phosphodiester DNA (PO-DNA) probes [19,32]
Summary
The promise of molecular tools that enable detection, regulation, and manipulation of genes, continues to inspire nucleic acid chemists to develop probes for sequence-unrestricted recognition of double-stranded DNA (dsDNA) [1,2,3,4,5]. We have recently presented an alternative approach, which is based on short DNA duplexes that are energetically activated for mixed-sequence recognition of dsDNA through installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′aminouridine monomer X or 2′-O-(pyren-1-yl)methyluridine monomer Y (Figure 1—for a definition of the zipper nomenclature, see the Experimental Section) [18,19] This structural motif forces the intercalating pyrene moieties into the same region of the duplex [20,21], leading to violation of the “nearest neighbor exclusion principle” [22,23] and destabilization of the duplex.
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