Serine-γPNA, Invader probes, and chimeras thereof: Three probe chemistries that enable sequence-unrestricted recognition of double-stranded DNA

Abstract

Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence in situ hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), i.e., single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with (R)-hydroxymethyl moieties, (ii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of 2’-O-(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, i.e., duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.

Graphical Abstract

Serine-γPNA, Invaders, and chimeras thereof, enable sequence-unrestricted recognition of chromosomal DNA under non-denaturing conditions.

INTRODUCTION.

Significant efforts continue to be devoted to the development of new approaches that enable sequence-specific recognition of double-stranded DNA (dsDNA). These efforts are motivated by the prospect of tools that facilitate regulation of gene expression at the transcriptional level, detection of diagnostic targets, and development of new drug modalities.

Early efforts focused on oligomers that recognize nucleobase-specific features from one of the nucleic acid duplex grooves, like triplex-forming oligonucleotides (TFOs)^1,2 and peptide nucleic acids (PNAs),^3,4 or pyrrole-imidazole polyamides.^5,6 However, these dsDNA-targeting probes have a limited target scope. Efficient triplex formation in the major groove generally requires the presence of long polypurine stretches. In contrast, minor-groove-binding polyamides are typically directed to short dsDNA regions as the recognition mechanism relies on binding- and shape-complementarity, which is compromised when regions longer than 6-8 base-pairs are targeted. While polyamides can be stitched together to target longer dsDNA regions,⁷ this increases their synthetic complexity.

These challenges spurred the development of strand-invading strategies, i.e., probes capable of unzipping the Watson-Crick base-pairs of dsDNA targets and forming new, more stable Watson-Crick base-pairs between probe strands and the complementary DNA (cDNA) regions. Examples include various modified single-stranded PNAs,^8–13 as well as double-stranded probes like pseudo-complementary PNAs,^14–18 Invader probes,¹⁹ heteroduplexes between intercalator-modified oligonucleotides and RNA, PNA or locked nucleic acid (LNA) strands,^20–22 and related approaches.^23–26 Some of the strand-invading strategies are discussed in more detail below.

γPNA, in which the electrostatically neutral and achiral N-(2-aminoethyl)glycine backbone of PNA is additionally modified at the γ-position with a (R)-methyl group, is an example of a single-stranded PNA probe with high cDNA affinity and strand-invading capabilities.⁸ To further increase solubility and reduce aggregation,²⁷ the γ-methyl group was replaced with a small hydrophilic (R)-diethylene glycol (mini-PEG or MP) moiety, resulting in chiral ^MPγPNA probes capable of sequence-unrestricted targeting of dsDNA duplexes under physiological conditions.^{9,28 MP}γPNA probes have been used to induce in vivo gene editing in a β-globin/eGFP mouse model,²⁹ detect telomeric DNA in human cell lines and tissues,³⁰ and identify bloodstream infections in whole blood.³¹

Serine-γPNAs (SγPNAs),³² having an l-serine side chain at the γ-position of the PNA backbone (Fig. 1), are easier to synthesize than ^MPγPNA and feature a hydrophilic hydroxymethyl moiety that also is expected to increase solubility and reduce aggregation. Although SγPNAs have been known for more than fifteen years and detection of miRNA in live cells³³ and gymnotic cellular uptake³⁴ has been demonstrated, their dsDNA-recognition characteristics are not well-characterized.

Figure 1.

Upper panel: Illustration of chromosomal DNA recognition using fluorophore-labeled single-stranded SγPNAs, double-stranded Invader probes, and chimeric SγPNA-Invader probes. Lower panel: structures of serine-γPNA and Invader building blocks.

Our laboratory has pursued the development of Invader probes,¹⁹ i.e., short DNA duplexes featuring one or more +1 interstrand zipper arrangements³⁵ of intercalator-modified nucleotides like 2′-O-(pyren-1-yl)methyl-RNA (Fig. 1). This monomer arrangement – termed an “energetic hotspot” – forces pairs of intercalating moieties into the same inter-base-pair region, resulting in a violation of the neighbor exclusion principle.³⁶ The principle asserts that local intercalator densities exceeding one intercalator per two base-pairs are energetically unfavorable in DNA duplexes due to limitations in local helix expandability (duplex is unwound by ~3.4 Å per intercalation event) and because stabilizing stacking interactions between neighboring base-pairs and the first intercalating moiety are perturbed.^37–39 Stated differently, intercalation is anti-cooperative at adjacent sites and, as a consequence, double-stranded Invader probes are partially unwound and destabilized.^40,41 The two probe strands, in turn, display high cDNA affinity as stabilizing stacking interactions between intercalators and flanking base-pairs ensue upon duplex formation (i.e., neighbor exclusion principle is no longer violated due to a lower local intercalator density). The greater stability of the double-stranded probe-target regions, vis-à-vis the double-stranded Invader probe and the dsDNA target region, provides the driving force for dsDNA-recognition via double-duplex strand invasion (Fig. 1).¹⁹ Invader probes have been shown to facilitate detection of (i) DNA fragments from specific food pathogens (28-mer mixed-sequence dsDNA fragments detected at 20 pM using Invader capture/signaling probes in sandwich assays),⁴² (ii) telomeric DNA of individual chromosomes in metaphasic spreads,⁴³ and (iii) gender-specific chromosomal-targets in interphase and metaphase nuclei under non-denaturing conditions.¹⁹

We recently evaluated the dsDNA-targeting characteristics of Invader and ^MPγPNA probes in a head-to-head study using a dsDNA model target comprising a partially self-complementary AT-rich region.⁴³ Invader probes were shown to result in more efficient and specific recognition in this sequence context while ^MPγPNAs were found to dimerize and be less available for dsDNA-recognition and/or to form mismatched duplexes.⁴³ These observations suggested that target regions with significant self-complementarity are challenging for single-stranded probes like ^MPγPNAs which have higher affinity towards themselves than DNA,⁴⁴ whereas such sequence contexts are less problematic for Invader probes that are engineered to form labile duplexes irrespective of the sequence context.

The shortcomings of ^MPγPNAs in partially self-complementary sequence contexts were to some degree rectified through hybridization with complementary Invader strands.⁴⁵ The ensuing double-stranded ^MPγPNA:Invader chimeric probes were found to be (i) relatively labile since PNA:DNA duplexes have insufficient flexibility to accommodate intercalators well,⁴⁶ and (ii) energetically activated for dsDNA-recognition since both probe strands exhibit high cDNA affinity. Thus, the mechanistic principles of ^MPγPNA:Invader chimeras resemble those of heteroduplexes between intercalator-modified oligonucleotides and RNA, PNA or locked nucleic acid (LNA) strands.^20–22 The chimeric ^MPγPNA:Invader probes resulted in more efficient and specific dsDNA-recognition than single-stranded ^MPγPNA probes.⁴⁵

In the present study we evaluated the dsDNA-recognition properties of SγPNA, Invader, and chimeric SγPNA:Invader probes, using DNA hairpin and chromosomal DNA targets with low levels of self-complementarity (Fig. 1).

RESULTS AND DISCUSSION

Probe design.

We evaluated the dsDNA-targeting properties of SγPNA, Invader, and chimeric SγPNA:Invader probes in sequence contexts with low self-complementarity. Towards this end, six SγPNAs were designed to target the sense and antisense strands of three different regions in a model target (Tables 1 and S1†), i.e., the DYZ-1 satellite gene⁴⁷ of the bovine (Bos taurus) Y chromosome (Fig. S1†). The SγPNAs target the same mixed-sequence regions as Invader probes INV2, INV4, and INV10 (Tables 1 and S1†), which were designed to have a modification density between 20% and 30% and were shown to recognize these DNA targets in preliminary non-denaturing fluorescence in situ hybridization experiments.⁴⁸ Access to these SγPNA and Invader strands also enabled the assembly of six chimeric SγPNA:Invader probes (Table 2).

Table 1.

Sequences of SγPNA and Invader probes studied herein, T_ms of probe duplexes and duplexes between individual probe strands and cDNA, and TA values.^a

		Tm [ΔTm] (°C)
Probes	Sequences	Probe duplex	5′-ON: cDNA	3′-ON: cDNA	TA (°C)
SγPNA2u	H-(TMR)-K-ATA CTG GTT TGT GTT C-K-NH2	-	>90 [>38.0]	-	>38.0
SγPNA2d	NH2-K-TAT GAC CAA ACA CAA G-K-(TMR)-H	-	-	>90 [>38.0]	>38.0
SγPNA4u	H-(TMR)-K-AGC CCT GTG CCC TG-K-NH2	-	65.0 [+4.5]	-	+4.5
SγPNA4d	NH2-K-TCG GGA CAC GGG AC-K-(TMR)-H	-	-	>90 [>29.5]	>29.5
SγPNA10u	H-(TMR)-K-GTG TAG TGT ATA TG-K-NH2	-	>90 [>47.0]	-	>47.0
SγPNA10d	NH2-K-CAC ATC ACA TAT AC-K-(TMR)-H	-	-	>90 [>47.0]	>47.0
INV2	5′-Cy3-AUA CUG GTT TGU GUT C-3′   3′-TAU GAC CAA ACA CAA G-Cy3-5′	39.0 [−13.0]	62.0 [+10.0]	69.0 [+17.0]	+40.0
INV4 b	5′-Cy3-AGC CCU GTG CCC TG-3′   3′-TCG GGA CAC GGG AC-Cy3-5′	61.5 [+1.0]	69.5 [+9.0]	75.5 [+15.0]	+23.0
INV10	5′-Cy3-GUG UAG TGU AUA TG-3′   3′-CAC AUC ACA UAU AC-Cy3–5′	46.0 [+3.0]	61.0 [+18.0]	67.0 [+24.0]	+39.0

^aΔT_m = change in T_m relative to the corresponding unmodified DNA duplexes, which are: DNA2 = 52.0 °C (reference for SγPNA2u, SγPNA2d, and INV2); DNA4 = 60.5 °C (reference for SγPNA4u, SγPNA4d, and INV4); DNA10 = 43.0 °C (reference for SγPNA10u, SγPNA10d, and INV10). Thermal denaturation curves were recorded in medium salt buffer ([Na⁺] = 110 mM, [Cl⁻] = 100 mM, pH 7.0 (NaH₂PO₄/Na₂HPO₄), [EDTA] = 0.2 mM) and [ON] = 1.0 μM. See main text for the definitions of TA. SγPNA probes are fully modified, while Invader strands are modified oligodeoxyribonucleotides. For structures of SγPNA and 2’-O-(pyren-1-yl)methyl-RNA A, C, and U monomers, see Fig. 1. K = lysine. TMR = TAMRA. For representative thermal denaturation profiles, see Figs. S5† and S6†.

^bData previously reported in reference 19.

Table 2.

Approximate T_ms and TAs for chimeric SγPNA:Invader probes.^a

Probes	Sequences	Approx. Tm (°C)	Approx. TA (°C)
SγPNA2u INV2d	H-(TMR)-K-ATA CTG GTT TGT GTT C-K-NH2     3′-TAU GAC CAA ACA CAA G-Cy3–5′	>80	>27
INV2u SγPNA2d	5′-Cy3-AUA CUG GTT TGU GUT C-3′ NH2-K-TAT GAC CAA ACA CAA G-K-(TMR)-H	>80	>20
SγPNA4u INV4d	H-(TMR)-K-AGC CCT GTG CCC TG-K-NH2     3′-TCG GGA CAC GGG AC-Cy3-5′	>90	<−10
INV4u SγPNA4d	5′-Cy3-AGC CCU GTG CCC TG-3′ NH2-K-TCG GGA CAC GGG AC-K-(TMR)-H	>90	-
SγPNA10u INV10d	H-(TMR)-K-GTG TAG TGT ATA TG-K-NH2     3′-CAC AUC ACA UAU AC-Cy3–5′	~75	>39
INV10u SγPNA10d	5′-Cy3-GUG UAG TGU AUA TG-3′ NH2-K-CAC ATC ACA TAT AC-K-(TMR)-H	~75	>33

^aΔT_m = change in T_m values relative to corresponding unmodified DNA duplexes, which are: DNA2 = 52.0 °C (reference for SγPNA2u:INV2d and INV2u:SγPNA2d); DNA4 = 60.5 °C (reference for SγPNA4u:INV4d and INV4u:SγPNA4d); and DNA10 = 43.0 °C (reference for SγPNA10u:INV10d and INV10u:SγPNA10d). Experimental conditions for T_m measurements are described in Table 1. TA (chimeric probe) = T_m (SγPNA:cDNA) + T_m (INV:cDNA) − T_m (chimeric probe duplex) − T_m (dsDNA). “-“ = cannot be calculated. For representative thermal denaturation profiles, see Fig. S8†.

Thermal denaturation properties.

Thermal denaturation temperatures (T_ms) were determined for (i) duplexes between SγPNA and cDNA, (ii) duplexes between individual Invader strands and cDNA, (iii) double-stranded Invader probes, and (iv) double-stranded chimeric SγPNA-Invader probes (Tables 1 and 2).

In agreement with prior reports,³² duplexes between SγPNA and cDNA were found to be exceedingly stable (T_ms >90 °C), except SγPNA4u:cDNA, which displayed a T_m of 65 °C (Table 1). The driving force for SγPNA-mediated dsDNA-recognition can be estimated by the “thermal advantage” (TA) given as TA = T_m (probe:cDNA) − T_m (dsDNA), with prominent positive values indicating a favorable driving force. Five single-stranded SγPNAs displayed favorable driving forces for dsDNA-recognition (TA >29.5 °C, Table 1). However, potential self-association of the SγPNAs must also be considered as it may impede dsDNA-recognition. Along these lines, we have previously observed that partially self-complementary ^MPγPNA probes self-associate, resulting in reduced dsDNA-targeting capacity.⁴³ Indeed, the thermal denaturation profiles of SγPNA2d, SγPNA4u, and SγPNA4d - in the absence of other strands - displayed sigmoidal transitions and considerable hyperchromicity, pointing to the formation of stable secondary structures (Fig. S7†). This was unexpected given the low self-complementarity of the sequence contexts studied herein as assessed by the IDT OligoAnalyzer^™ Tool.⁴⁹ Reliable T_m determination was not feasible as significant hysteresis was observed for these transitions. In contrast, SγPNA2u, SγPNA10u, and SγPNA10d displayed minimal hyperchromicity and irregular, if any, sigmoidal transitions (Fig. S7†). The underlying reasons for the different denaturation profiles of the SγPNAs are unclear.

As expected, the three double-stranded Invader probes were found to be relatively labile (T_ms = 39.0-61.5 °C, Table 1), whereas duplexes between individual Invader strands and cDNA were more stable (T_ms = 61.0-75.5 °C, Table 1). These differences reflect that the neighbor exclusion principle is violated in the double-stranded Invader probes (high local intercalator density) but not in duplexes between individual Invader strands and cDNA (lower local intercalator density). The driving force for Invader-mediated dsDNA-recognition can be estimated as TA = T_m (5′-ON:cDNA) + T_m (3′-ON:cDNA) − T_m (probe duplex) − T_m (dsDNA). The Invader probes are substantially activated for dsDNA-recognition as evidenced by the prominent TA values (TA = 23.0-43.0 °C, Table 1). The lower TA value observed for INV4 likely reflects its less modified nature.⁴⁸

The chimeric duplexes between complementary SγPNA and Invader strands were unexpectedly stable, with T_ms ranging from ~75 °C to more than 90 °C (Table 2). However, the irregular nature of the denaturation profiles indicates that distorted duplex geometries are adopted (Fig. S8†). Most of the chimeric probes displayed a favorable driving force for dsDNA-recognition (TA >20 °C, Table 2).

Spectroscopic characterization of chimeric probes.

UV-Vis absorption spectra were recorded for individual Invader strands and the corresponding duplexes with complementary DNA, SγPNA, and Invader strands to gain insight into the structural factors that underlie the observed thermal denaturation trends (Fig. S9†). Individual Invader strands display pyrene absorption maxima in the 333-334 nm and 348-350 nm range. Hybridization with cDNA results in average bathochromic shifts of ~3.3 nm of the pyrene absorption maximum at ~350 nm (Tables 3 and S2†), which is consistent with pyrene intercalation.⁵⁰ Less pronounced bathochromic shifts were observed upon hybridization with complementary SγPNA or Invader strands (~0.5 and ~0.7 nm, respectively, Tables 3 and S2†), which is indicative of less prevalent pyrene intercalation.

Table 3.

Average shifts of pyrene absorption maxima in the ~350 nm region and average I₅/I₁ fluorescence emission intensity ratios upon hybridization of single-stranded Invader probes with complementary DNA, SγPNA, or single-stranded Invader (ssINVs) probes.^a

Average Δλmax (nm)			Average I5/I1 ratio
+cDNA	+SγPNA	+ssINV	+cDNA	+SγPNA	+ssINV
3.3	0.5	0.7	1.25	1.08	1.27

^aDatasets used to calculate these average values are shown in Tables S2† and S3†.

Steady-state fluorescence emission spectra (λ_ex = 350 nm) of these duplexes display typical I₁ and I₅ pyrene emission peaks at ~377 and ~397 nm (Fig. S10†). The I₅/I₁ intensity ratio is known to be dependent on the polarity of the environment that the pyrene moieties are located in with higher values reflecting a more hydrophobic micro-environment.⁵¹ Chimeric probes display lower I₅/I₁ intensity ratios than Invader:cDNA or Invader duplexes (average values: ~1.08 vs ~1.25 and ~1.27, respectively, Tables 3 and S3†). These observations are consistent with our interpretations of the results from the UV-Vis experiments, in as much they suggest that the pyrene moieties of the chimeric SγPNA-Invader probes are located in a more polar environment (i.e., not intercalated) than those of Invader:cDNA duplexes (i.e., intercalated). This also provides a rationale for the lower T_ms observed for SγPNA:Invader vis-à-vis SγPNA:cDNA duplexes as placement of hydrophobic intercalators in the polar grooves is expected to be less favorable.

Recognition of mixed-sequence DNA targets – initial screen.

The dsDNA-targeting properties of SγPNA, Invader, and chimeric SγPNA-Invader probes were evaluated using an electrophoretic mobility shift assay (EMSA) in which the probes are incubated with corresponding 3′-digoxigenin (DIG)-labeled DNA hairpin (DH) model targets.¹⁹ Thus, DH2, DH4, and DH10 are comprised of double-stranded stems that are iso-sequential relative to the corresponding probes (Fig. 2 and Table S4†). Each stem is linked at one end by a decameric thymidine loop, resulting in high-melting hairpins (T_ms = 72, 62, and 82 °C for DH2, DH4, and DH10, respectively, Table S4†), which reduces the potentiality of strand fraying. Successful recognition of a DNA hairpin is expected to result in the formation of a ternary complex (Fig. 2), manifesting as a slower-moving band relative to the DNA hairpin when incubation mixtures are resolved via non-denaturing polyacrylamide gel electrophoresis (nd-PAGE).

Figure 2.

Assay used to evaluate recognition of DNA hairpin model targets by single-stranded SγPNA, double-stranded Invader probes, or double-stranded chimeric SγPNA-Invader probes.

Complete recognition was observed when a 100-fold molar excess of SγPNA2d, SγPNA10u, or SγPNA10d were incubated with their respective DNA hairpin targets for 2.5 h at 37 °C (Fig. 3). This is consistent with the favorable driving force for dsDNA-recognition noted for these probes (TA >38 °C, >47 °C, and >47 °C, respectively, Table 1). SγPNA4u and SγPNA4d also resulted in recognition of DH4 (~34% and >90%, respectively), but prominent band fading, indicative of probe aggregation and low solubility,⁹ was observed (Fig. 3). Consistent with this, band fading was far less pronounced when a lower excess of SγPNA4u or SγPNA4d was used (Figs. S11† and S13†). Inexplicably, given the favorable TA value and absence of self-association, minimal recognition was observed with SγPNA2u (<10%, Fig. 3). Extending incubation to 15 hours did not result in further dsDNA-recognition, indicating that recognition with single-stranded SγPNAs is complete within 2.5 h (compare Figs. S11† and S12†).

Figure 3.

Representative electrophoretograms from recognition experiments in which a 100-fold molar excess of different SγPNA or Invader probes were incubated with their respective model dsDNA hairpin targets DH2, DH4, and DH10. Histograms depict averaged results from at least three experiments with error bars representing standard deviation. RC = band corresponding to recognition complex. DH = band corresponding to DNA hairpin. DIG-labeled DH2, DH4, and DH10 (sequences shown in Table S4†) were incubated with 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) at 37 °C for 2.5 h. DH2 = target for SγPNA2u, SγPNA2d, and INV2; DH4 = target for SγPNA4u, SγPNA4d, and INV4; DH10 = target for SγPNA10u, SγPNA10d, and INV10.

Invader probes INV2, INV4, and INV10 resulted in ~60%, <5%, and >90% recognition of the corresponding hairpin targets when used at a 100-fold molar excess and incubated for 2.5 h (Fig. 3). When incubation was extended to 15 h, near-complete recognition was also observed for INV2, while recognition with INV4 remained minimal (Figs. S14† and S15†; also shown later in Fig. 5). The lack of hairpin recognition seen with INV4 is likely related to its higher stability and/or less favorable driving force (T_m = 61.5 °C and TA = 23 °C, Table 1).

Figure 5.

Binding specificity of SγPNA, Invader, and chimeric SγPNA-Invader probes. An excess of probe (25-fold molar excess for SγPNA and chimeric SγPNA-Invader probes; 100-fold molar excess for Invader probes) was incubated with corresponding DNA hairpins featuring stems of identical sequence or differing in sequence at one (“m”) or two positions (“mm”) relative to the probes. For sequences of DNA hairpins, see Table S4†. Gel electrophoretograms depicting binding between chimeric SγPNA-Invader probes and matched DNA hairpins at these conditions are shown in Fig. S17†. Incubation conditions are as described in Figure 3 except for incubation times of 2.5 h (SγPNA) or 15 h (Invader and chimeric SγPNA-Invader probes).

Chimeric probes SγPNA2d:INV2u, SγPNA10u:INV10d, and SγPNA10d:INV10u resulted in complete recognition of the corresponding hairpin targets when incubated at 100-fold molar excess at 37 °C for 2.5 h, just as the SγPNA-only counterparts (Fig. 4). Interestingly, SγPNA2u:INV2d resulted in ~50% recognition (Fig. 4), whereas the single-stranded SγPNA2u only resulted in trace recognition (Fig. 3); this, suggests that recognition of the second DNA hairpin arm by INV2d drives additional recognition. No target recognition was observed for chimeric probes SγPNA4u:INV4d and SγPNA4d:INV4u, presumably due to their high stability (T_m >90 °C) and/or unfavorable driving force (TA_{SγPNA4u:INV4d} <−10 °C).

Figure 4.

Representative electrophoretograms from recognition experiments in which a 100-fold molar excess of different chimeric SγPNA:Invader probes were incubated with their respective model dsDNA hairpin targets DH2, DH4, and DH10 for 2.5 h at 37 °C. Incubation conditions are as described in Figure 3. Binding partners are as follows: DH2 = target for SγPNA2u:INV2d and INV2u:SγPNA2d; DH4 = target for SγPNA4u:INV4d and INV4u:SγPNA4d; DH10 = target for SγPNA10u:INV10d and INV10u:SγPNA10d.

Recognition of mixed-sequence DNA targets – dose-response curves.

Dose-response relationships were determined for SγPNA, Invader, and chimeric SγPNA:Invader probes to elaborate on the results from the initial screen (Figs. S13†–S19†). Thus, C₅₀ values - i.e., the probe concentrations resulting in 50% recognition of the DNA hairpin targets - of ~0.12, ~0.35, and ~0.25 μM were observed for SγPNA2d, SγPNA10u, and SγPNA10d, respectively, following 2.5 hours of incubation (Table 4). The corresponding Invader probes INV2 and INV10 display C₅₀ values of ~1.9 and ~1.6 μM following 2.5 hours of incubation, and C₅₀ values of ~0.16 and ~0.21 μM following 15 hours of incubation (Table 4). Thus, INV2 and INV10 result in similar levels of recognition following 15 hours of incubation as SγPNA2d, SγPNA10u, and SγPNA10d after 2.5 hours of incubation. Chimeric probes SγPNA2u:INV2d, SγPNA2d:INV2u, SγPNA10u:INV10d and SγPNA10d:INV10u display similar C₅₀ values following 15 hours of incubation as the corresponding SγPNA and Invader probes (0.13-0.22 μM, Table 4), whereas slightly higher C₅₀ values were observed when incubation was reduced to 2.5 hours (0.17-0.58 μM, Table 4).

Table 4.

Summary of C₅₀ values for SγPNA, Invader, and chimeric SγPNA:Invader probes following 2.5 or 15 hours of incubation.^a

Probe	C50, 2.5h (μM)	C50, 15h (μM)
SγPNA2u	-c	-c
SγPNA2d	0.12	-b
SγPNA4u	-d	-d
SγPNA4d	-d	-d
SγPNA10u	0.35	-b
SγPNA10d	0.25	-b
SγPNA2u:INV2d	0.58	0.20
SγPNA2d:INV2u	0.17	0.20
SγPNA4u:INV4d	-c	-c
SγPNA4d:INV4u	-c	-c
SγPNA10u:INV10d	0.33	0.22
SγPNA10d:INV10u	0.38	0.13
INV2	1.9	0.16
INV4	-c	-c
INV10	1.6	0.21

^aC₅₀ values were determined from the dose-response curves shown in Figs. S18† and S19†.

“-“ = not determined due to:

^bfast recognition kinetics,

^clow levels of recognition in initial screen, or

^dband fading.

Thus, each of the three probe chemistries allows for efficient recognition of DH2 and DH10 in this assay. The slower binding kinetics of the double-stranded probes vis-à-vis single-stranded SγPNAs, likely reflect the need of the former to undergo partial denaturation for dsDNA-recognition to ensue. Similar observations were made with Invader probes vis-à-vis single-stranded ^MPγPNAs.⁴³ It is interesting that Invader probes are more strongly affected by shorter incubation times than the chimeric SγPNA:Invader probes, given the lower stability of the Invader probes (compare probe T_ms, Tables 1 and 2). We speculate that the chimeric SγPNA:Invader probes adopt geometries that, while more stable, are irregular and more conducive to dsDNA-recognition.

Recognition of model mixed-sequence dsDNA targets – binding specificity.

The binding specificity of the SγPNA, Invader, and chimeric SγPNA:Invader probes was evaluated by incubating them with DNA hairpins featuring stems that differ in sequence at one or two positions relative to the probes (Fig. 5 and Table S4†).

DNA hairpins differing in stem sequence at two positions were fully discriminated by SγPNA2d, SγPNA10u, and SγPNA10d. In contrast, incubation with complementary hairpin targets resulted in complete recognition at the experimental conditions employed (25-fold molar excess, 2.5 h incubation, Fig. 5). However, DNA hairpins differing in sequence at only one position were incompletely discriminated. Thus, SγPNA2d led to full recognition of DH2m, whereas SγPNA10u and SγPNA10d resulted in ~50% and trace recognition of DH10m, respectively (Fig. 5). SγPNA2u, SγPNA4u, and SγPNA4d, did not result in recognition of complementary or mismatched DNA hairpins (Fig. S20†). When used at a higher (100-fold) molar excess, SγPNA2d, SγPNA10u, and SγPNA10d resulted in near-complete recognition of the singly mismatched hairpins (>90%), while SγPNA2d and SγPNA10d also resulted in moderate recognition (~30%) of the doubly mismatched hairpins (Fig. S21†). Furthermore, SγPNA4u and SγPNA4d resulted in moderate recognition of singly mismatched hairpin DH4m, and minor recognition of the doubly mismatched hairpin DH4mm (Fig. S21†). To sum up, some SγPNA probes allow for efficient recognition of dsDNA targets but only with moderate binding fidelity.

Remarkably, INV2 and INV10 fully discriminated the corresponding singly and doubly mismatched DNA hairpins even when used at 100-fold molar excess and incubated for 15 hours (Fig. 5). Similarly, chimeric SγPNA-Invader probes SγPNA2u:INV2d, SγPNA10u:INV10d, and SγPNA10d:INV10u fully discriminated singly and doubly mismatched DNA hairpins when used at 25-fold molar excess (Fig. 5), i.e., conditions resulting in substantial-to-full recognition of the complementary DNA hairpin (Fig. S17†). Only SγPNA2d:INV2u resulted in substantial (~70%) recognition of the singly mismatched DNA hairpin DH2m (Fig. 5). Even when used at 100-fold molar excess, SγPNA10u:INV10d and SγPNA10d:INV10u displayed near-perfect discrimination of singly and doubly mismatched DNA hairpins, whereas SγPNA2d:INV2u did not discriminate (>90% recognition) the singly mismatched DNA hairpin (Fig S22†). Thus, all of the Invader probes and chimeric SγPNA-Invader probes - with the exception of SγPNA2d:INV2u - display similar binding affinity towards dsDNA and improved binding specificity vis-à-vis the corresponding single-stranded SγPNA probes.

The improved specificity of the double-stranded Invader and chimeric SγPNA-Invader probes is likely due to stringency clamping effects,^15,52 as binding to non-target DNA hairpins would necessitate dissociation of a double-stranded probe and the formation of recognition complexes with two mismatched and energetically destabilized duplexes.⁵³

Detection of chromosomal DNA.

Next, we evaluated the dsDNA-targeting properties of the TAMRA-labeled SγPNA, Cy3-labeled Invader, and TAMRA/Cy3-labeled chimeric SγPNA-Invader probes in the context of nd-FISH experiments. As mentioned before, the probes were designed to target three different regions in the bovine Y chromosome (~6 x 10⁴ tandem repeats of ~1175 bp, Fig. S1†). The probes were incubated with isolated fixed nuclei from a male bovine kidney cell line under otherwise non-denaturing conditions and visualized by fluorescence microscopy. Successful target recognition was expected to manifest itself in the form of a single punctate fluorescent signal in G1-phase interphase nuclei or two closely spaced signals in S/G2-phase interphase nuclei.¹⁹

Invader probes INV2, INV4, and INV10 resulted in efficient binding to the target regions as indicated by the observation that >85% of interphase nuclei displayed a single, prominent punctate signal against a low background (Fig. 6). The signaling capacity of INV4 vis-à-vis INV2 and INV10 is counterintuitive given the lack of hairpin-recognition (Fig. 3). We attribute this observation to the higher target region copy number for INV4 (six per tandem repeat vs. one per repeat for INV2 and INV10) and/or the presence of a non-canonical target geometry that facilitates INV4 binding.⁴⁸ The lack of signals when INV2, INV4, or INV10 were incubated with isolated nuclei from a female bovine endothelial cell line, strongly suggests that these probes bind in a target-specific manner (Fig. S35†).

Figure 6.

Representative images of INV2, INV4, INV10, SγPNA2d, SγPNA10d, SγPNA4u:INV4d, and SγPNA10d:INV10u probes incubated with fixed nuclei from a male bovine kidney cell line (3 h, 37 °C, Tris-Cl/EDTA buffer, pH 8.0). Images are overlays of Cy3/TAMRA (red) and DAPI channels (blue). Scale bar denotes ~10 μm. Probes were incubated at the following concentrations: 12.5 nM Invaders (INV4 used at 1/4^th concentration), 5.0 nM single-stranded SγPNA, or 2.5 nM chimeric probes).

SγPNA2d and SγPNA10d resulted in the formation of single punctate signals in ~70% of interphase nuclei when used at a concentration of 5 nM (Fig. 6; see also Figs. S24† and S28† and Table S5†). However, a stronger non-specific background was observed than with the Invader probes. The remaining SγPNA probes resulted in no or minimal target-specific recognition when used at a concentration of 5 nM; instead, multiple signals and/or intense diffuse background were observed (Figs. S23† and S25†–S27† and Table S5†). The use of higher probe concentrations (12.5 nM) resulted in an increase of non-specific signals with all SγPNA probes except for SγPNA2d, for which a slightly higher proportion of single-signal nuclei (~80%) was observed (Figs. S23†–S28† and Table S5†). No signals were observed when SγPNA2d or SγPNA10d were incubated with isolated nuclei from the female bovine endothelial cell line (Fig. S36†).

Chimeric SγPNA-Invader probes resulted in the formation of single punctate signals in only ~30-60% of interphase nuclei when used at a concentration of 2.5 nM, and a significant proportion of nuclei displayed multiple, non-specific signals and/or high background (Fig. 6; see also Figs. S29†–S34† and Table S5†). When SγPNA4u:INV4d or SγPNA10d:INV10u were used at a concentration of 6.25 nM, formation of single punctate signals was observed in ~80% and ~60% of the nuclei respectively, whereas all other chimeric SγPNA-Invader probes yielded extensive non-specific signals (Figs. S29†–S34†).

Thus, despite the three probe types displaying comparable binding affinities towards DNA hairpin targets, and double-stranded Invader and chimeric probes displaying improved discrimination of mismatched DNA hairpins, marked differences were observed in the nd-FISH experiments. Invader probes resulted in the formation of specific signals with the three targets studied, whereas only some of the SγPNA and chimeric SγPNA-Invader probes exhibited acceptable signal characteristics, typically against a more intense non-specific background. While the reasons for the different trends in the DNA hairpin and nd-FISH experiments are not fully understood, it should be noted that the experimental conditions (e.g., buffers, probe concentrations) are quite different, which may impact probe binding.

CONCLUSION.

Three dsDNA-targeting probe chemistries have been evaluated, i.e., single-stranded serine-γPNAs, double-stranded Invader probes, and double-stranded chimeric SγPNA:Invader probes, in a model mixed-sequence dsDNA target with low self-complementarity. SγPNAs were found to display exceptional cDNA-affinity, some propensity for formation of stable secondary structures and/or aggregates, and efficient and fast recognition of model mixed-sequence dsDNA hairpin targets (for 4-5 of six probes) but only with moderate specificity. Invader probe duplexes were found to be comparatively labile, whereas the individual probe strands displayed moderately high cDNA affinity, resulting in a favorable driving force for dsDNA recognition. Recognition of model mixed-sequence dsDNA hairpin targets was as efficient and more specific than with SγPNA probes (for two of three probes) but slower, requiring longer incubation times. The chimeric SγPNA:Invader probes were found to be surprisingly stable yet to display a favorable driving force for dsDNA-recognition (for 3–4 of six probes) due to the high cDNA-affinity of the individual probe strands. Recognition of the model mixed-sequence dsDNA hairpin targets was as efficient as with SγPNA and Invader probes (for four of six probes) and proceeded with intermediate specificity and kinetics. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, albeit to varying degrees. The Invader probes were found to display the most promising and consistent characteristics (single punctate signals in >90% of interphase nuclei against a low background and no signal in negative control experiments), whereas two of six SγPNA and two of six chimeric SγPNA-Invader probes displayed acceptable characteristics (single punctate signals in 60%-80% of interphase nuclei against a higher background). Recognition of GC-rich target regions proved particularly challenging for SγPNA and chimeric SγPNA-Invader probes. The findings disclosed herein expands our knowledge of dsDNA-targeting probe chemistries, which will aid the design of future experiments and biomedical applications.

EXPERIMENTAL SECTION

Synthesis and purification of SγPNA strands.

Boc-protected serine γ-PNA monomers (A, T, C, G) were purchased from a commercial vendor (ASM Chemicals and Research, Hanover, Germany). SγPNAs were synthesized via solid-phase synthesis using a MBHA (4-methylbenzhydrylamine) resin. The monomers were dissolved in a mixture of 0.2 M of 1-methyl-2-pyrrolidone (NMP, Sigma Aldrich), 0.52 M N,N-diisopropylethylamine, and 0.39 M HBTU, and activated for ~3 min. The TAMRA dye (Biotium) was conjugated to the N-terminus of PNAs. Following synthesis, SγPNAs were cleaved off from the resin using a cleavage cocktail containing m-cresol, thioanisole, trifluoromethanesulfonic acid (TFMSA) and trifluoroacetic acid (TFA) (1:1:2:6, v/v/v/v), followed by precipitation using diethyl ether. SγPNAs were purified and characterized by RP-HPLC (Shimadzu) using an acetonitrile (containing 0.1% trifluoroacetic acid) and water (containing 0.1% trifluoroacetic acid) based solvent system (Figs. S2† and S3†). MALDI–TOF spectrometry (performed at Tuft Univ.) was used to characterize the synthesized SγPNAs (Table S1†). The concentration of SγPNAs was estimated using the following extinction coefficients at 260 nm (ε₂₆₀) of individual monomers: A (13,700 M⁻¹cm⁻¹), C (6,600 M⁻¹cm⁻¹), G (11,700 M⁻¹cm⁻¹), and T (8,600 M⁻¹cm⁻¹).

Synthesis and purification of Invader strands.

The Invader strands used herein, i.e., ONs modified with 2’-O-(pyren-1-yl)methyl-RNA monomers A, C, and U, were prepared and purified as previously described.^54,55 The purity and identity of the synthesized ONs was verified by analytical ion-pair reverse phase HPLC (XTerra MS C18 column: 0.05 M triethyl ammonium acetate and acetonitrile gradient) and MALDI-MS analysis recorded on a Quadrupole Time-of-Flight (Q-TOF) mass spectrometer using 3-hydoxypicolinic acid as a matrix (Table S1†). Concentrations of Invader strands were estimated using the following extinction coefficients (OD₂₆₀/μmol): G (12.01), A (15.20), T (8.40), C (7.05), pyrene (22.40)⁵⁶ and Cy3 (4.93)⁵⁷.

Thermal denaturation experiments.

Thermal denaturation temperatures of duplexes were measured on a Cary 100 UV-Vis spectrophotometer equipped with a 12-cell Peltier temperature controller and determined as the maximum of the first derivative of thermal denaturation curves (A₂₆₀ vs. T) recorded in medium salt buffer (T_m buffer: 100 mM NaCl, 0.2 mM EDTA, and pH 7.0 adjusted with 10 mM Na₂HPO₄ and 5 mM Na₂HPO₄) using each strand at 1.0 μM concentration. Strands were mixed in quartz optical cells with a path-length of 1.0 cm and annealed by heating to 85 °C (30 min), followed by slow cooling (2 °C/min) to the starting temperature, and equilibration (30 min) prior to initiation of the experiments. The temperature of the denaturation experiments ranged from at least 15 °C below the T_m to at least 15 °C above the T_m (though not above 98 °C). A temperature ramp of 0.5 °C/min was used, resulting in minimal hysteresis for Invader probes (Fig. S5†), Invader:cDNA duplexes (Fig. S5†), and SγPNA:cDNA duplexes (Fig. S6†) (T_ms determined from heating and cooling cycles differed by less than 2.5 °C). Pronounced hysteresis was observed for single-stranded SγPNAs, rendering determination of their T_m values unreliable (Fig. S7†). Denaturation curves of SγPNA:Invader duplexes were irregularly sigmoidal (Fig. S8†) consistent with distorted duplex geometries, rendering determination of T_m values challenging. Where possible, T_ms are reported as averages of at least two experiments within ±1.0 °C.

Optical spectroscopy.

UV-Vis absorption spectra (range 200–600 nm) were recorded at 10 °C using the same samples (i.e., each strand at 1.0 μM in T_m buffer) and instrumentation as in the thermal denaturation experiments. Steady-state fluorescence emission spectra were recorded using a Cary Eclipse fluorimeter in non-deoxygenated T_m buffer to mimic the conditions of bioassays (each strand used at 1.0 μM) and obtained as an average of five scans. An excitation wavelength of λ_ex = 350 nm and scan speed of 600 nm/min were used along with excitation and emission slits of 5.0 nm and 2.5 nm, respectively. Experiments were performed at 5 °C under N₂ flow to ascertain maximal hybridization of probes.

Electrophoretic mobility shift assays.

The electrophoretic mobility shift assays were performed as previously described.¹⁹ Thus, DNA hairpins were obtained from commercial sources and used without further purification. Hairpins were digoxigenin (DIG)-labeled using the oligonucleotide 3′-end labeling procedure provided with the 2^nd Generation DIG Oligonucleotide 3′-End Labeling Kit (Roche Applied Biosciences) as recommended by the manufacturer. Briefly, 11-digoxigenin-ddUTP was incorporated at the 3′-end of the hairpin (100 pmol) using a recombinant terminal transferase. The reaction mixture was quenched by addition of EDTA (0.05 M), diluted to 68.8 nM, and used without further processing. Invader or SγPNA probes (concentration as specified) were heated (90 °C for 2 min) and then cooled to room temperature (over ~30 min), and subsequently incubated with the specified DIG-labeled DNA hairpin (final concentration 34.4 nM) in HEPES buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl₂, pH 7.2, 10% sucrose, 1.44 mM spermine tetrahydrochloride (the latter two are included to mimic the nuclear viscosity and protein environment)) at 37 °C for the specified time. Following incubation, loading dye (6x) was added and the mixtures were loaded onto 16% non-denaturing TBE-PAGE gels (45 mM tris-borate, 1 mM EDTA; acrylamide:bisacrylamide, 19:1)). Electrophoresis was performed using constant voltage (~70 V) at ~4 °C for ~1.5 h. Bands were then blotted onto positively charged nylon membranes (~100 V, 30 min, ~4 °C) and cross-linked through exposure to UV light (254 nm, 5 × 15 W bulbs, 5 min). Membranes were incubated with anti-digoxigenin-alkaline phosphatase F_ab fragments as recommended by the manufacturer and transferred to a hybridization jacket. Membranes were incubated with the chemiluminescence substrate (CSPD) for 10 min at 37 °C, and chemiluminescence of the formed product was captured on X-ray films. Digital images of developed X-ray films were obtained using a BioRad ChemiDoc MP imaging system, which was also used for quantification of the bands. The percentage of hairpin recognition was calculated as the intensity ratio between the recognition complex band and unrecognized hairpin. An average of three independent experiments is reported with standard deviations (±). Non-linear regression analysis was used to fit data points from dose-response experiments. A script written for the “Solver” module in Microsoft Office Excel,⁵⁸ was used to fit following equation to the data: y = C + A(1 − e^−kt) where C, A and k are constants. The resulting equation was used to calculate C₅₀ values by setting y = 50 and solving for t.

Cell culture and nuclei preparation.

Male bovine kidney cells (MDBK (NLB-1), ATCC: CCL-22, Bethesda, MD) were maintained in DMEM with GlutaMax (Gibco, 10569-010) and 10% fetal bovine serum (Invitrogen). Female bovine endothelial cells (CPAE, ATCC: CCL-209) were maintained in Eagle’s Minimum Essential Medium (ATTC, 30-2003) and 20% fetal bovine serum (Invitrogen). Cells were cultured in separate 25 mL or 75 mL flasks at 37.5 °C in a 5% CO₂ atmosphere for 72-96 h to achieve 70-80% confluency. At this point, colcemid (Gibco KaryoMax, 15210-040) (65 μL per 5 mL of growth media) was added and the cells were incubated at 37.5 °C and 5% CO₂ for two more hours. The medium was then replaced with a 37.5 °C solution of 0.05% Trypsin-EDTA in DMEM to detach adherent cells. The cell suspension was transferred to a centrifuge tube and centrifuged (10 min, 1000 rpm) to pellet the cells. The supernatant was discarded, and the dislodged cell pellet was incubated with a hypotonic 75 mM KCl solution (5–8 mL, 20 min, 23 °C), followed by addition of fixative (10 drops, MeOH:AcOH, 3:1, v/v) and further incubation with gentle mixing (10 min, room temperature). The suspension was centrifuged (10 min, 1000 rpm), the supernatant discarded, and additional fixative solution (5-8 mL) added to the nuclei suspension, which was followed by gentle mixing and incubation (30 min, room temperature). The centrifugation/resuspension/incubation with fixative solution steps were repeated three additional times. The final pellet – containing somatic nuclei – was resuspended in the fixative solution and used immediately or stored at 4 °C for up to two weeks.

Preparation of slides for FISH experiments.

The nuclei suspension was resuspended in fresh fixative solution. Glass microscope slides were dipped in distilled water to create a uniform water layer across the slide. An aliquot of the nuclei suspension (3-5 μL or enough to cover the slide) was dropped onto the slide, while holding the slide at a 45° angle, and allowed to run down the length of the slide. Slides were then allowed to dry at a ~20° angle in an environmental chamber at 28 °C and a relative humidity of 38% to fix nuclei and evaporate solvents.

Fluorescence in situ hybridization.

An aliquot of the labelling solution consisting of Cy3-labeled Invader, TAMRA-labeled SγPNA or Cy3/TAMRA-labeled SγPNA:Invader chimeric probes in deionized water (100 μL) and PCR buffer (100 μL, 10 mM Tris-HCl, 50 mM KCl, pH 8.3) was dropped on each slide. Slides were placed in a glass culture dish, covered with a lid, and incubated at 37 °C for ~3 h). Slides were washed in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) for 3 min at 37 °C by submersion and gentle pipetting, rinsed with autoclaved water by pipetting, and allowed to dry at room temperature. Once dried, Gold SlowFade Plus DAPI (3 μL, Invitrogen) was added on each slide and a round glass coverslip was mounted and sealed with clear nail polish. A Nikon Eclipse Ti-S/L100 inverted fluorescence microscope, equipped with a SOLA SMII LED light source system and Cy3/TAMRA and DAPI filter cubes, was used to visualize nuclei at 100x magnification. Images of fluorophore-labelled nuclei were captured using a 14-bit Cool SNAP HQ2 cooled CCD camera and processed with NIS Elements BR 4.20 imaging software. The qualitative assessment of signal strength was determined through comparison of the signal brightness of all counted nuclei (on average ~100 nuclei were counted per probe).