Nicked Invader probes: multistranded and sequence-unrestricted recognition of double-stranded DNA

Abstract

Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for enabling tools for applications in molecular biology, diagnostics, and medicine. Towards this end, we have previously introduced Invader probes, i.e., short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. The individual strands of these labile probes display high affinity towards complementary DNA (cDNA), which drives sequence-unrestricted dsDNA-recognition. However, recognition of long targets is challenging due to the high stability of the corresponding probes. To address this, we recently introduced toehold Invader probes, i.e., Invader probes with 5'-single-stranded overhangs. The toehold architecture allows for shorter double-stranded segments to be used, which facilitates probe dissociation and dsDNA-recognition. As an extension thereof, we here report the biophysical and dsDNA-targeting properties of nicked Invader probes. In this probe architecture, the single-stranded overhangs of toehold Invader probes are hybridized to short intercalator-modified auxiliary strands, leading to formation of additional labile segments. The extra binding potential from the auxiliary strands imparts nicked Invader probes with greater dsDNA-affinity than the corresponding toehold or blunt-ended probes. Recognition of chromosomal DNA targets, refractory to recognition by conventional Invader probes, is demonstrated for nicked Invader probes in the context of non-denaturing FISH experiments, which highlights their utility as dsDNA-targeting tools.

Figure 1.

Schematic of the dsDNA-recognition process using a) blunt-ended (conventional), b) toehold, or c) nicked Invader probes. d) Structure of 2’-O-(pyren-1-yl)methyl RNA monomer used to generate energetic hotspots.

Figure 2.

a) Sequences of nicked Invader probes used herein. Main and auxiliary strands are shown in black and red, respectively. The corresponding toehold Invader probes are comprised of main strands only. U = 2′-O-(pyren-1-yl)methyluridine. C = 2′-O-(pyren-1-yl)methylcytidine monomers. b) Illustration of a representative nicked Invader probe and its three duplex segments.

Figure 3.

a) Illustration of the electrophoretic mobility shift assay used to evaluate dsDNA-recognition of Invader probes. b) Representative gel electrophoretograms from recognition experiments in which DH1 was incubated with a 50-fold molar excess of different probes. RC = recognition complex. c) Histogram depicting averaged results from at least three independent experiments with error bars representing standard deviation. Conditions: DIG-labeled DH1 (50 nM) incubated with a 50-fold molar excess of the specified probe in HEPES buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl₂, pH 7.2, 10% sucrose, 1.44 mM spermine tetrahydrochloride) for 17 h at 37 °C. The sequence of DH1 is shown in Table S2†. The electrophoretogram is a composite image from different runs.

Figure 4.

Dose-response profiles for recognition of DH1 by nicked Invader probes NIP1-NIP4 at 37 °C. Profiles are constructed based on the electrophoretograms shown in Figs. S29 and S30†. Experimental conditions are as described in Fig. 3.

Figure 5.

Binding specificity of nicked Invader probe NIP2. (a) Illustration of the mismatched recognition complexes that would ensue upon recognition of MM1-MM3 by NIP2; arrows indicate the position of mismatched base-pairs. For sequences of MM1-MM3, see Table S2†. Equivalent illustrations for recognition of MM1-MM3 with NIP1, NIP3 and NIP4 are shown in Fig. S33†. (b) Representative electrophoretograms from experiments in which NIP2 was incubated with non-complementary targets MM1-MM3 or complementary target DH1. For equivalent electrophoretograms entailing NIP1, NIP3 and NIP4, see Fig. S33†. Pre-annealed 3’-DIG-labelled hairpins (50 nM) were incubated with a 50-fold molar excess of pre-annealed probe at 37 °C for 17 h in HEPES buffer as outlined in Fig. 3. The electrophoretogram is a composite image from different runs.

Figure 6.

Representative gel electrophoretograms from recognition experiments in which a 50-fold molar excess of different DYZ-1-targeting probes was incubated with complementary target DH2 or non-complementary target DH2-MM at 37 °C for 17 h. For sequences of DH2 and DH2-MM and structures of recognition complexes formed, see Table S2† and Fig. S34†, respectively. Experimental conditions are as described in Fig. 2. The electrophoretogram is a composite image from different runs.

Figure 7.

Images from nd-FISH experiments using (a) nicked Invader probe DYZ-NIP (1 ng per 200 μl of PCR buffer, 3 h, 37.5 °C) or (b) conventional Invader probe DYZ-REF (6 ng, 3 h, 37.5 °C). Fixed isolated interphase nuclei from male bovine kidney cells were incubated with probes in a Tris buffer (20 mM Tris-Cl, 100 mM KCl, pH 8.0) and counterstained with DAPI. Images were obtained by overlaying images from Cy3 (red) and DAPI (blue) channels and adjusting the exposure. Nuclei were viewed at 60X magnification using a Nikon Eclipse Ti-S inverted microscope. Probe concentrations were selected based on initial optimization studies (Fig. S35†).