Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes

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.

a For structure of monomers X and Y see Figure 1 in the main manuscript. Figure S1. Representative thermal denaturation profiles of duplexes between X-/Y-modified PS-DNA strands and cDNA. For experimental conditions, see Table 1. Table S2. DNA selectivity of X-and Y-modified PS-DNA a .

Binding Specificity of X-/Y-modified PS-DNA
The binding specificities of centrally modified 9-mer PS-DNA strands were studied using DNA targets with mismatched nucleotides opposite to the modification (Table S3). Excellent discrimination of the C-mismatched target is observed, while discrimination of G-or T-mismatched targets is much less efficient. On the other hand, doubly modified 9-mer PS-DNA discriminate DNA targets with a single mismatched nucleotide opposite of the central 2′-deoxyriboadenosine very efficiently (Table S4). These trends mirror our observations with X-/Y-modified PO-DNA strands, further suggesting that the pyrene moieties are intercalating upon cDNA hybridization [1,2].     Figure S2. Absorption spectra of X1-X6 in absence or presence of cDNA/cRNA. Spectra were recorded at T = 5 °C using each strand at 1.0 μM concentration in Tm buffer. Note, different axis scales are used. Figure S3. Absorption spectra of Y1-Y6 in absence or presence of cDNA/cRNA. Spectra were recorded at T = 5 °C using each strand at 1.0 μM concentration in Tm buffer. Note, different axis scales are used.   , serves as a first approximation to describe the energy difference between the 'products' and 'reactants' of the prototypical recognition process, with more positive values signifying greater thermodynamic dsDNA recognition potential [3]. See Table 1 in main manuscript for T m 's of 5′-Inv:cDNA and 3′-Inv:cDNA. See Table 2 in the main manuscript for T m 's of Invader probes. T m of the isosequential dsDNA target is 37.5 °C [3]. b Data from reference [3].

Protocol-Stability of Invader Probes against DNase 1
An aqueous solution of DNase I (Worthington Biochemical Corporation-0.61 μL of a 2 μg/mL solution) was added to a 6.3 μM solution of a specific pre-annealed Invader in TE buffer (100 μL, 10 mM Tris·HCl, 0.1 mM EDTA, 10 mM MgCl2, pH 8.0) and the mixture was incubated at 20 °C in a water bath. Aliquots (10 μL) were removed at specific times (0.5, 1, 2, 5, 10, 20 and 30 min) and degradation was quenched by addition of ethidium bromide buffer (2.0 mL, 5 mM Tris·HCl, 0.5 mM EDTA, 0.5 μg/mL EtBr, pH 8.0). The fluorescence intensity of the solution was measured (λex = 525 nm; λem = 600 nm) using the same instrumentation employed for the steady-state fluorescence experiments. Intensities were averaged over 15 minutes. Experiments were performed in duplicates and representative graphs are shown.

Discussion-Stability of Invader Probes against DNase 1
The stability of Invader probes against DNase I was evaluated using an ethidium bromide based assay. In this assay, high levels of fluorescence are observed when the studied duplex is intact due to intercalating ethidium bromide, while low levels of fluorescence are expected if a duplex has been degraded to single strands (or shortened to a level where the duplex dissociates) [4,5].
Similar assays have been used to show that long PS-DNA duplexes exhibit excellent stability against DNase I [6,7]. Figure S7 shows the fluorescence intensity profiles of representative 13-mer Invaders in the presence of DNase I. The PO-DNA analogs of X13:X14 and Y13:Y14 are moderately resistant to DNase I degradation with half-lives of 15 min and >30 min, respectively, whereas the unmodified PO-DNA duplex is rapidly degraded.
PS-DNA Invaders X13:X14 and Y13:Y14 did not show any change in fluorescence emission relative to background EtBr buffer (data not shown), presumably because the probe duplexes are dissociated into single strands at the experimental conditions used for this assay (Tm of X13:X14 and Y13:Y14 are <25 °C, Table 2).