Chimeric γPNA–Invader probes: using intercalator-functionalized oligonucleotides to enhance the DNA-targeting properties of γPNA

Raymond G. Emehiser and Patrick J. Hrdlicka *
Department of Chemistry, University of Idaho, Moscow, ID-83844, USA. E-mail: hrdlicka@uidaho.edu

Received 24th December 2019 , Accepted 9th January 2020

First published on 13th January 2020


Gamma peptide nucleic acids (γPNAs), i.e., single-stranded PNA strands that are modified at the γ-position with (R)-diethylene glycol, and Invader probes, i.e., DNA duplexes with +1 interstrand zipper arrangements of 2′-O-(pyren-1-yl)methyl-RNA monomers, are two types of nucleic acid mimics that are showing promise for sequence-unrestricted recognition of double-stranded (ds) DNA targets. We recently demonstrated that recognition of dsDNA targets with self-complementary regions is challenging for single-stranded high-affinity probes like γPNAs due to their proclivity for secondary structure formation, but not so for Invader probes, which are engineered to form readily denaturing duplexes irrespective of the target sequence context. In the present study, we describe an approach that mitigates these limitations and improves the dsDNA-recognition properties of γPNAs in partially self-complementary target contexts. Chimeric probes between γPNAs and individual Invader strands are shown to form metastable duplexes that (i) are energetically activated for recognition of complementary mixed-sequence dsDNA target regions, (ii) reduce γPNA dimerization, and (iii) substantially improve the fidelity of the dsDNA-recognition process. Chimeric γPNA–Invader probes are characterized with respect to thermal denaturation properties, thermodynamic parameters associated with duplex formation, UV-Vis and fluorescence trends to establish pyrene binding modes, and dsDNA-recognition properties using DNA hairpin model targets.


Introduction

Considerable efforts have been dedicated to the development of chemically modified oligonucleotides and nucleic acid mimics that enable sequence-specific recognition of double-stranded DNA (dsDNA) targets. These efforts are fueled by the promise for tools that can be used to regulate gene expression, detect diagnostically relevant targets, and edit disease-related mutations. Pioneering approaches, such as triplex-forming oligonucleotides and peptide nucleic acids (PNAs),1,2 recognize base-pair-specific features from the major groove of DNA duplexes but are limited to recognition of extended polypurine regions. Hence, alternative strategies have been explored to realize sequence-unrestricted dsDNA-recognition.3–16 Among these, the so-called miniPEG-γPNA (γPNA)17,18 and Invader probes19 present themselves as particularly promising approaches (Fig. 1 and 2).
image file: c9ob02726b-f1.tif
Fig. 1 Structures of modifications and nucleic acid mimics used in this study.

image file: c9ob02726b-f2.tif
Fig. 2 Illustration of recognition of dsDNA by single-stranded γPNA or LNA probes, and double-stranded Invader and chimeric probes.

γPNAs, in which the sugar-phosphate backbone of DNA is replaced with an electrostatically neutral N-(2-aminoethyl)glycine backbone that is additionally modified at the γ-position with a small hydrophilic (R)-diethylene glycol (miniPEG) moiety (Fig. 1), display very high affinity towards complementary DNA (cDNA).17 The increased cDNA-affinity of γPNAs is due to favorable strand pre-organization caused by the chirality-inducing γ-substituent, which also serves to increase aqueous solubility and reduce aggregation. This provides the driving force for recognition of mixed-sequence dsDNA targets via duplex invasion (Fig. 2).18,20 Accordingly, γPNAs have been used to induce in vivo gene editing in a β-globin/eGFP mouse model,21 detect telomeric DNA in human cell lines and tissues,22 and identify bloodstream infections in whole blood.23

Invader probes are short DNA duplexes that are modified with one or more +1 interstrand zipper arrangements of intercalator-modified nucleotides like 2′-O-(pyren-1-yl)methyl-RNA (Fig. 1). This probe architecture forces two intercalating pyrene moieties to compete for the same inter-base-pair space (Fig. 2), resulting in localized unwinding and destabilization of the probe24,25 as the neighbor exclusion principle26 – which asserts that intercalators bind to a maximum loading of one intercalator per two base pairs27 – is violated.

In contrast, the two strands of an Invader probe display very high affinity towards their respective cDNA targets as duplex formation results in reduced intercalator loading and strongly stabilizing stacking interactions between intercalators and flanking base-pairs. The difference in stability between the Invader probe and dsDNA target region relative to the duplexes between individual Invader probe strands and cDNA provides the driving force for recognition of complementary mixed-sequence dsDNA regions via double-duplex invasion (Fig. 2). We have demonstrated that Invader probes can recognize (i) DNA fragments specific to different food pathogens (28-mer mixed-sequence dsDNA fragments were detected at 20 pM with excellent binding specificity in a sandwich assay using Invader capture/signaling probes),28 (ii) target regions on Y-chromosomes in interphase and metaphase nuclei from male bovine kidney cells under non-denaturing conditions,29 and (iii) telomeric DNA of individual chromosomes in metaphasic spreads with excellent specificity.30

We recently evaluated Invader and γPNA probes in a common model system, in which the dsDNA target comprised a partially self-complementary AT-rich region.30 Invader probes were shown to result in substantially more efficient and specific recognition than γPNAs in this sequence context, as the latter were found to dimerize and form partially complementary duplexes across the AT-rich region of the non-target strand. These observations underscored that targets with high levels of self-complementarity are challenging for single-stranded high-affinity probes like γPNAs, whereas this target context is less problematic for Invader probes, which are engineered to form predictable and easily denaturing duplexes, irrespective of the sequence context of the target region.

In the present study, we set out to develop an approach that mitigates these limitations for γPNAs in such sequence contexts. Our strategy towards this end entails chimeric probes between γPNAs and individual Invader strands (Fig. 2). The approach is inspired by a recent study in which duplexes between conventional PNA strands and complementary oligodeoxyribonucleotides (ONs) with a high content of ethynylperylene-modified D-threoninol bulges were shown to recognize mixed-sequence dsDNA targets with moderate efficiency under heat-shock conditions.31 The reported approach is based on the observation that PNA:DNA duplexes do not appear to have sufficient flexibility to accommodate intercalators,32 resulting in readily denaturing probe duplexes. Since the PNA and perylene-modified ON strands display moderately increased cDNA affinity, a modest dsDNA-recognition driving force ensues for these probes. In related approaches, easily denaturing probe duplexes between pyrene-modified ONs and complementary RNA33 or locked nucleic acid (LNA)34 strands have also shown potential for recognition of mixed-sequence dsDNA targets since intercalators are more poorly accommodated in the A-type probe duplexes vis-à-vis B-type probe:cDNA duplexes.35

In exploring double-stranded chimeric probes between single-stranded Invader strands (ssINV) and complementary γPNA or LNA strands (Fig. 2), we hypothesized that the probes not only will denature readily and be strongly activated for mixed-sequence dsDNA-recognition via double-duplex invasion, but also reduce dimerization of γPNA and LNA strands and improve the specificity of the recognition process.

Results and discussion

Experimental design and thermal denaturation properties

A series of Invader, γPNA, and LNA strands – originally prepared in connection with our recent study30 – were used to assemble eight chimeric γPNA–Invader and LNA–Invader probes (Table 1). Access to these strands also enabled comparison to a set of control probes (Table S1) including the corresponding conventional Invader probes with three +1 interstrand zipper arrangements of X monomers – a design known to yield an optimal balance between binding affinity and binding specificity in this sequence context30 – and single-stranded γPNA and LNA probes. The γPNA strands are fully modified and lysine-capped to improve aqueous solubility, while the LNA strands are modified ONs with an evenly distributed LNA monomer content of ∼31%. This modification pattern and density is known to result in prominent increases in melting temperatures (Tms) per modification (higher LNA densities result in saturation effects).36 The probes were designed to target the partially self-complementary 13-mer AT-rich dsDNA segment mentioned before, which we have used for evaluation of different Invader probe designs.29,30
Table 1 Sequences of probes studied herein, Tms of probe duplexes and duplexes between individual probe strands and cDNA, and TA valuesa
  T mTm] (°C)  
Entry Probes Sequences Probe duplex Upper strand vs. cDNA Lower strand vs. cDNA TA (°C)
a LNA monomers are denoted by lower case letters (“c” = 5-methylcytosine LNA monomer). ΔTm = change in Tm relative to the unmodified DNA duplex, Tm (5′-GGTATATATAGGC[thin space (1/6-em)]:[thin space (1/6-em)]3′-CCATATATATCCG) = 37.5 °C. For a definition of TA, see the main text. Buffer composition: [Na+] = 110 mM, [Cl] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4) using 1.0 μM of each strand. Data in “upper strand vs. cDNA” and “lower strand vs. cDNA” columns are from ref. 30.
1 γPNA1 H-Lys-GGT ATA TAT AGG C-Lys-NH2 75.0 [+37.5] >85 [>47.5] 62.5 [+25.0] >35.0
ssINV2 3′-CCA [X with combining low line]AT A[X with combining low line]A [X with combining low line]CC G
2 ssINV1 5′-GG[X with combining low line] ATA [X with combining low line]A[X with combining low line] AGG C 59.5 [+22.0] 61.5 [+24.0] 79.0 [+41.5] +43.5
γPNA2 NH2-Lys-CCA TAT ATA TCC G-Lys-H
3 γPNA1 H-Lys-GGT ATA TAT AGG C-Lys-NH2 77.0 [+39.5] >85 [>47.5] 63.0 [+25.5] >33.5
ssINV4 3′-CCA [X with combining low line]A[X with combining low line] ATA [X with combining low line]CC G
4 ssINV3 5′-GG[X with combining low line] A[X with combining low line]A TA[X with combining low line] AGG C 58.0 [+20.5] 61.0 [+23.5] 79.0 [+41.5] +44.5
γPNA2 NH2-Lys-CCA TAT ATA TCC G-Lys-H
5 LNA1 5′-GgT AtA TaT AgG C 73.0 [+35.5] 54.0 [+16.5] 62.5 [+25.0] +6.0
ssINV2 3′-CCA [X with combining low line]AT A[X with combining low line]A [X with combining low line]CC G
6 ssINV1 5′-GG[X with combining low line] ATA [X with combining low line]A[X with combining low line] AGG C 72.0 [+34.5] 61.5 [+24.0] 55.0 [+17.5] +7.0
LNA2 3′-CCa TAt ATa TCc G
7 LNA1 5′-GgT AtA TaT AgG C 66.5 [+29.0] 54.0 [+16.5] 63.0 [+25.5] +13.0
ssINV4 3′-CCA [X with combining low line]A[X with combining low line] ATA [X with combining low line]CC G
8 ssINV3 5′-GG[X with combining low line] A[X with combining low line]A TA[X with combining low line] AGG C 62.0 [+24.5] 61.0 [+23.5] 55.0 [+17.5] +16.5
LNA2 3′-CCa TAt ATa TCc G


T ms were determined for double-stranded probes and duplexes between individual probe strands and cDNA (Tables 1 and S1). As reported,30 conventional Invader probes are more labile than duplexes between individual Invader strands and cDNA with Tms of 51–52 °C versus Tms of 61–63 °C (entries 1 and 2, Table S1), respectively. Although quite stable, chimeric γPNA–Invader probes display Tms that are 8–21 °C lower than the corresponding γPNA:cDNA duplexes (compare Tms of 58–77 °C for probe duplexes with Tms > 79 °C for γPNA:cDNA duplexes, entries 1–4, Table 1). This supports our hypothesis that γPNA strands display reduced affinity towards complementary Invader strands. In contrast, chimeric LNA–Invader probes are much more stable than the corresponding duplexes between LNA-modified ONs and cDNA (compare Tms of 62–73 °C for probe duplexes with Tms of 54–55 °C for LNA:cDNA duplexes, entries 5–8, Table 1). These results indicate that the pyrene moieties of the Invader strands adopt different binding modes depending on the nature of their binding partner.

Thermodynamic driving force for recognition of complementary dsDNA targets

The driving force for recognition of complementary dsDNA targets by double-stranded probes can be approximated by the term thermal advantage, which we define as TA = Tm (upper strand vs. cDNA) + Tm (lower strand vs. cDNA) − Tm (probe duplex) − Tm (dsDNA) (Tables 1 and S1). Alternatively, the available free energy for recognition of complementary dsDNA by double-stranded probes at 310 K can be parameterized as ΔG310rec = ΔG310 (upper probe vs. cDNA) + ΔG310 (lower probe vs. cDNA) − ΔG310 (probe) − ΔG310 (dsDNA) (Tables 2 and S2; for a detailed background discussion of the TA and ΔG310rec terms, see ref. 30). For single-stranded probes, these terms reduce to TA = Tm (probe vs. cDNA) − Tm (dsDNA), which is simply the ΔTm value for a probe:cDNA duplex, and ΔG310rec = ΔG310 (probe vs. cDNA) − ΔG310 (dsDNA), which is the ΔΔG310 value for a probe:cDNA duplex.
Table 2 Change in Gibbs free energy at 310 K (ΔG310) upon formation of chimeric probe duplexes, and duplexes between individual probe strands and cDNA. Also shown is the calculated change in reaction free energy upon probe-mediated recognition of isosequential dsDNA targets (ΔG310rec)a
  ΔG310 [ΔΔG310] (kJ mol−1)  
Entry Probes Sequences Probe duplex Upper strand vs. cDNA lower strand vs. cDNA ΔG310rec (kJ mol−1)
a ΔΔG310 is determined relative to the corresponding unmodified DNA duplex (ΔG310 = −42 kJ mol−1). For a definition of ΔG310rec, see the main text. For enthalpic and entropic parameters, see Tables S3 and S4.† nd = not determined due to unclear baseline. For experimental conditions, see Table 1. Data for entries in “upper strand vs. cDNA” and “lower strand vs. cDNA” columns are from ref. 30.
1 γPNA1 H-Lys-GGT ATA TAT AGG C-Lys-NH2 −59 [−17] nd −76 [−34] nd
ssINV2 3′-CCA [X with combining low line]AT A[X with combining low line]A [X with combining low line]CC G
2 ssINV1 5′-GG[X with combining low line] ATA [X with combining low line]A[X with combining low line] AGG C −58 [−16] −80 [−38] −109 [−67] −89
γPNA2 NH2-Lys-CCA TAT ATA TCC G-Lys-H
3 γPNA1 H-Lys-GGT ATA TAT AGG C-Lys-NH2 −71 [−30] nd −78 [−37] nd
ssINV4 3′-CCA [X with combining low line]A[X with combining low line] ATA [X with combining low line]CC G
4 ssINV3 5′-GG[X with combining low line] A[X with combining low line]A TA[X with combining low line] AGG C −61 [−19] −78 [−36] −109 [−67] −84
γPNA2 NH2-Lys-CCA TAT ATA TCC G-Lys-H
5 LNA1 5′-GgT AtA TaT AgG C −75 [−34] −63 [−21] −76 [−34] −22
ssINV2 3′-CCA [X with combining low line]AT A[X with combining low line]A [X with combining low line]CC G
6 ssINV1 5′-GG[X with combining low line] ATA [X with combining low line]A[X with combining low line] AGG C −92 [−51] −80 [−38] −68 [−26] −11
LNA2 3′-CCa TAt ATa TCc G
7 LNA1 5′-GgT AtA TaT AgG C −88 [−46] −63 [−21] −78 [−37] −12
ssINV4 3′-CCA [X with combining low line]A[X with combining low line] ATA [X with combining low line]CC G
8 ssINV3 5′-GG[X with combining low line] A[X with combining low line]A TA[X with combining low line] AGG C −75 [−33] −78 [−36] −68 [−26] −29
LNA2 3′-CCa TAt ATa TCc G


Thermodynamic parameters associated with duplex formation were determined by baseline fitting of thermal denaturation curves (Tables 2 and S2–S4).37 Chimeric γPNA–Invader probes are very strongly activated for dsDNA-recognition (ΔG310rec < −84 kJ mol−1, entries 1–4, Table 2), which, in largest part, is due to the high stability of probe:cDNA duplexes (ΔΔG310 between −67 and −34 kJ mol−1 per duplex), whereas the moderate stability of the probe duplexes (ΔΔG310 between −30 and −16 kJ mol−1) partially reduces the driving force. Of note, chimeric γPNA–Invader probes display a greater driving force for dsDNA-recognition than single-stranded γPNA probes or conventional Invader probes (ΔG310rec values between −67 and −60 kJ mol−1, entries 1–4, Table S2). This is particularly interesting considering that the calculated ΔG310rec values do not take potential self-dimerization of the single-stranded γPNA probes into account (ΔG310 approx. −10 kJ mol−1);30 partially dimerized γPNA probes must be denatured as part of the dsDNA-recognition process, which further reduces the dsDNA-targeting driving force.

The chimeric LNA–Invader probes are, by comparison, far less thermodynamically activated for dsDNA-recognition (ΔG310rec values between −29 and −11 kJ mol−1, entries 5–8, Table 2). This is largely due to the high stability of the probe duplexes (i.e., ΔΔG310 between −51 and −33 kJ mol−1), which largely offsets the moderately stabilizing contributions from LNA:cDNA and Invader:cDNA duplexes (i.e., ΔΔG310 values between −21 and −38 kJ mol−1). In fact, the driving force for dsDNA-recognition using chimeric LNA–Invader probes is generally less favorable than with single-stranded LNA probes (ΔG310rec values between −26 and −21 kJ mol−1, entries 5 and 6, Table S2).

Thus, the thermodynamic driving force for recognition of complementary dsDNA regions decreases in the following order: γPNA–Invader probes > conventional Invader probes ≥ single-stranded γPNA probes ≫ single-stranded LNA probes ≥ LNA-Invader probes. Similar conclusions are reached upon analysis of the corresponding Tm-based TA term (for a discussion, see ESI).

Optical spectroscopic characterization

UV-Vis absorption and steady-state fluorescence emission spectra were recorded for single-stranded Invader probes and the corresponding duplexes with complementary DNA, γPNA, LNA, and Invader strands to gain further insight into the structural underpinnings of the observed thermal denaturation and thermodynamic trends (Fig. 3, S2 and S3). For example, hybridization-mediated intercalation of pyrene moieties is known to induce bathochromic shifts of pyrene absorption bands, whereas hypsochromic shifts are observed if pyrene moieties are moved towards the minor groove.38
image file: c9ob02726b-f3.tif
Fig. 3 Representative (a) UV-vis and (b) steady-state emission spectra for ssINV2 and the corresponding duplexes with complementary DNA, γPNA, LNA, and Invader strands. Each strand was used at 1.0 μM in Tm buffer. T = 10 °C (UV-Vis) or 5 °C (fluorescence). λex = 350 nm. For full dataset, see Fig. S2 and S3.

As expected, two main pyrene absorption peaks are observed in the 330–355 nm region (Fig. 3a). In agreement with observations from our prior studies,25 pyrene absorption bands are shifted bathochromically when individual Invader strands are hybridized with cDNA, whereas hypsochromic shifts are observed upon formation of double-stranded Invader probes as pyrene-nucleobase stacking is perturbed (average Δλmax = 1.3 nm and −3.3 nm, respectively, Table 3). Prominent hypsochromic shifts are observed upon formation of chimeric γPNA–Invader probes, whereas minor bathochromic shifts are observed upon formation of chimeric LNA–Invader probes (average Δλmax = −5.8 nm and 1.3 nm, respectively, Table 3). These results clearly indicate that the position of the pyrene moieties of an Invader strand vary depending on the nature of the binding partner.

Table 3 Average shifts of pyrene absorption bands in the 345–360 nm region for single-stranded Invader probes upon hybridization with complementary DNA, γPNA, LNA or Invader strandsa
Average Δλmax (nm)
+cDNA +γPNA +LNA +ssINV
a “±” denotes standard deviation. Measurements were performed at 10 °C in Tm buffer using quartz optical cells with a 1.0 cm path length. For full dataset, see Table S5.†
1.3 ± 1.5 −5.8 ± 3.3 1.3 ± 1.5 −3.3 ± 2.0


Steady-state fluorescence emission spectra for chimeric γPNA–Invader probes display typical I1 and I5 pyrene emission peaks at ∼377 nm and ∼397 nm (Fig. 3b and S3). Formation of these duplexes is accompanied by an overall increase in emission and substantially reduced I5/I1 intensity ratios39vis-à-vis single-stranded Invader strands (I5/I1 = ∼0.9 vs. ∼1.4, respectively, Table S6). This, along with the observed Tm and thermodynamic trends and the hybridization-induced hypsochromic shifts of pyrene absorption bands, is consistent with reduced pyrene-nucleobase interactions40 and localization of the pyrene moieties in a polar extrahelical environment,25,41 presumably the minor groove as this is the positional preference for O2′-fluorophore-functionalized ribonucleotides in A-type duplexes.42 Only minor changes in emission (Fig. 3b and S3), yet prominent I5/I1 ratios (∼1.2, Table S6), indicative of pyrene intercalation into the hydrophobic duplex core, are observed upon formation of chimeric LNA–Invader probes. Formation of conventional Invader probes does not result in substantial emission changes in the pyrene monomer region but does result in the emergence of an intense and broad peak centered around ∼500 nm, which is consistent with a pyrene-pyrene excimer (Fig. 3b). As discussed in our earlier reports,24,25 positioning of 2′-O-(pyren-1-yl)methyl-RNA monomers in +1 interstrand zipper arrangements forces the pyrene moieties to intercalate (I5/I1 = ∼1.5, Table S6), where they compete for the same hydrophobic inter-base-pair region, thus increasing the likelihood for pyrene-pyrene stacking and excimer emission. ssINV2 also shows significant excimer emission, presumably due to partial pyrene-mediated probe aggregation.

Recognition of model mixed-sequence dsDNA targets – design and initial screen

The dsDNA-targeting properties of the chimeric γPNA–Invader and LNA–Invader probes, and the various control probes were characterized using an established electrophoretic mobility shift assay,29 in which the probes are incubated with a digoxigenin (DIG)-labelled DNA hairpin (DH) model target. DH1 comprises a 13-mer dsDNA target region that is isosequential vis-à-vis the probes and which is linked at one end by a decameric thymidine loop (Fig. 4). Recognition of DH1 is expected to manifest itself by the appearance of slower-moving bands when incubation mixtures are resolved by non-denaturing polyacrylamide gel electrophoresis (nd-PAGE).
image file: c9ob02726b-f4.tif
Fig. 4 Recognition of a DNA hairpin model target by chimeric γPNA–Invader, conventional Invader, or single-stranded γPNA, LNA or Invader probes.

We previously demonstrated that incubation of DH1 with a 5-fold molar excess of γPNA1 results in ∼59% recognition at 37 °C (lane 6, Fig. 5), whereas no recognition is observed with γPNA2 (lane 7).30 We have attributed the lack of recognition with γPNA2 to probe dimerization, which occurs due to the partially self-complementary nature of the probe.30γPNA1 also dimerizes but displays higher cDNA affinity and an additional binding mode, which facilitates dsDNA-recognition.30 Thus, the slowest-moving recognition band formed upon incubation of DH1 with γPNA1 (lane 6), represents a ternary complex in which two γPNA1 strands are bound to DH1 in anti-parallel orientation, i.e., with one γPNA1 strand binding to the complementary stem strand and a second γPNA1 strand – unexpectedly – binding across the central 8-mer AT-region of the non-target strand with single-stranded γPNA-G overhangs stabilizing this duplex through end-capping on either side (see also Fig. S4).30 We stipulate that a similar binding mode is precluded for γPNA2 due to its lover cDNA affinity and the fact that the equivalent 8-mer duplex with the non-target strand would feature less stabilizing γPNA-C overhangs.


image file: c9ob02726b-f5.tif
Fig. 5 Representative electrophoretograms for incubation mixtures between model dsDNA target DH1 and a 5-fold molar excess of various probes. Histograms depict averaged results from at least three experiments with error bars representing standard deviation. Complex = recognition complex(es). DIG-labeled DH1 (34.4 nM, 5′-GGTATATATAGGC-T10-GCCTATATATACC-3′) was 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. All recognition bands formed were considered when quantifying the dsDNA-targeting efficiency of γPNA1, γPNA1:ssINV2 and γPNA2:ssINV1.

Gratifyingly, improved DH1 recognition is observed when chimeric γPNA–Invader probes are used vis-à-vis single-stranded γPNA probes. Thus, γPNA1:ssINV2 results in ∼67% recognition of DH1 with no traces of the ternary DH1:(γPNA1)2 complex (lane 2, Fig. 5),43 presumably since hybridization of ssINV2 to its complementary stem target is more favorable than potential unintended binding of γPNA1 to the central 8-mer AT-region of the non-target strand (see also Fig. S4). Even more remarkably, γPNA2:ssINV1 results in ∼54% recognition of DH1 (lane 3, Fig. 5), whereas no recognition is observed with γPNA2 (lane 7). We submit that recognition-impeding γPNA2 dimerization is minimized by the metastable nature of the γPNA2:ssINV1 probe, thus facilitating DH1-recognition. Equivalent trends are observed for γPNA1:ssINV4 and γPNA2:ssINV3 probes (lanes 2 and 3, Fig. S5). As expected,18 control duplex γPNA1:γPNA2 does not result in recognition of DH1 (lane 4, Fig. 5) since the γPNA duplex is exceedingly stable (Tm >85 °C, results not shown). These results suggest that the use of labile chimeric γPNA–Invader probes may alleviate the problems observed when single-stranded γPNAs are designed to target dsDNA regions of a highly self-complementary nature.

Conventional Invader probes ssINV1:ssINV2 and ssINV3:ssINV4 result in very efficient recognition of DH1 (∼74% and ∼77%, respectively, lane 5 in Fig. 5 and lane 5 in Fig. S5), whereas individual Invader strands only result in minimal recognition of DH1 (lanes 8 and 9 in Fig. 5 and lanes 8 and 9 in Fig. S5).

Chimeric LNA–Invader probes were also evaluated but only result in trace recognition of DH1 (<10%, Fig. S6), as anticipated from the only moderately favorable ΔG310rec values (Table 2). Chimeric LNA–Invader probes were, therefore, not evaluated further.

Recognition of model mixed-sequence dsDNA targets – dose–response, time–course and binding specificity studies

Next, dose–response experiments were performed for a representative subset of probes to determine C50 values, i.e., the probe concentrations resulting in 50% recognition of DH1 (Fig. 6a and S7–S9). Chimeric γPNA–Invader probes display C50 values in the 45–165 nM range, whereas the single-stranded γPNA1 probe displays a substantially higher C50 value of 250 nM (Table 4). The single-stranded γPNA2 probe does not result in discernible recognition of DH1 even when used at 200-fold molar excess (Fig. S7). These results further substantiate the hypothesis that dsDNA-recognition characteristics of γPNA probes can be improved through formation of metastable duplexes with complementary Invader strands.
image file: c9ob02726b-f6.tif
Fig. 6 (a) Dose–response and (b) time–course curves for recognition of DH1 using a subset of chimeric γPNA–Invader probes and single-stranded γPNA probes. Incubation conditions are as described in Fig. 5 except that variable probe concentrations or incubation times are used. For corresponding electrophoretograms, see Fig. S7 and S10. Data for γPNA1 have been previously reported in ref. 30. Bars denote standard deviations.
Table 4 Summary of dsDNA-recognition parameters for a representative subset of chimeric γPNA–Invader, conventional Invader, and single-stranded γPNA probesa
Probes C 50 (nM) Rec5X (%) t 50 (min)
a Rec5X = level of DH1-recognition using 5-fold molar probe excess (standard deviation listed). C50 and t50 determined from Fig. 6, S5 and S9.† Data for γPNA1, ssINV1:ssINV2 and ssINV3:ssINV4 have been previously reported in ref. 30. nd = not determined.
γPNA1 250 59 ± 5 <10
γPNA1:ssINV2 77 67 ± 9 <10
γPNA2:ssINV1 122 54 ± 7 108
γPNA1:ssINV4 45 59 ± 8 nd
γPNA2:ssINV3 164 47 ± 4 nd
ssINV1:ssINV2 44 74 ± 14 68
ssINV3:ssINV4 48 77 ± 13 37


Interestingly, chimeric γPNA–Invader probes display C50 values that rival those of conventional Invader probes (Table 4), highlighting the potential of these probes for applications relying on sequence-unrestricted recognition of dsDNA.44 The efficient DNA-recognition seen for conventional Invader probes vis-à-vis chimeric γPNA–Invader probes is surprising at first given that the driving force of the latter is greater (e.g., compare ΔG310rec values for entries 1–2 and 1–4, Tables S2 and 2, respectively). However, conventional Invader probes denature more readily, which likely lowers the activation barrier for dsDNA-recognition (e.g., compare ΔG310 values for probe duplexes, entries 1–2 and 1–4, in Tables S1 and 2, respectively).

Recognition kinetics were evaluated for a subset of chimeric γPNA–Invader probes (Fig. 6b and S10) and compared to previously reported data for γPNA1 and conventional Invader probes.30 Interestingly, γPNA1 and γPNA1:ssINV2 result in rapid recognition of DH1 (t50 < 10 min, Table 4) whereas recognition using γPNA2:ssINV1 (t50 ∼108 min, Table 4) or conventional Invader probes is substantially slower (t50 = 37–68 min, Table 4). The slower binding kinetics observed for γPNA2:ssINV1 and conventional Invader probes are not surprising given that these double-stranded probes likely must undergo partial denaturation as part of the dsDNA recognition process. The comparatively faster recognition kinetics seen for γPNA1 are expected for a single-stranded probe that recognizes complementary dsDNA targets via duplex invasion. However, the fast binding kinetics seen for the γPNA1:ssINV2 probe are puzzling given the observed Tm and ΔG310 trends for γPNA1:ssINV2 and γPNA2:ssINV1 (compare values for entries 1 and 2 in Tables 1 and 2). The faster recognition kinetics for γPNA1:ssINV2 are likely related to the more beneficial dsDNA-recognition properties displayed by γPNA1 vis-a-vis γPNA2. We speculate that recognition of DH1 using the chimeric γPNA1:ssINV2 probe is facilitated by rapid formation of the ternary DH1:(γPNA1)2 complex, which is rapidly transformed into the final DH1:γPNA1:ssINV2 ternary complex (Fig. S11).

When analysing the recognition kinetics of the probes studied herein, it is important to recognize that the DNA hairpin model targets constitute a different target context than e.g., PCR fragments (100s–1000s of base-pairs; relaxed B-type DNA), plasmids (1000s of base-pairs; supercoiling), or chromosomal DNA (millions of base-pairs; histone-association), for which different recognition kinetics should be expected.

Lastly, the binding specificity of a subset of chimeric γPNA–Invader and single-stranded γPNA probes was evaluated. Thus, a 5-fold molar probe excess was incubated at 37 °C with DIG-labelled DNA hairpins DH2DH7 (Table S7), which have fully base-paired stems that differ in sequence at either the 6- or 9-position vis-à-vis the probes (Fig. 7). As previously reported,30γPNA1 displays minimal discrimination of these singly mismatched dsDNA targets. This contrasts previous observations in which γPNA were shown to fully discriminate a 291 bp dsDNA with centrally located single mismatches.18 We speculate that the difference in observations is due to the use of different assays, incubation conditions, and target contexts (i.e., long relaxed dsDNA with internal target regions vis-à-vis DNA hairpins, in which the target regions are integrated in the stem region).


image file: c9ob02726b-f7.tif
Fig. 7 Binding specificity of representative chimeric γPNA:Invader and γPNA probes against non-complementary targets DH2–DH7. Representative electrophoretograms from experiments in which a 5-fold molar probe excess was incubated with DH1–DH7 (34.4 nM) at 37 °C for 2.5 h. Conditions are as described in Fig. 5. Lanes have been rearranged to facilitate presentation but are from the same electrophoretogram. Data for γPNA1 from ref. 30.

In contrast, the chimeric γPNA–Invader (Fig. 7) and conventional Invader probes (Fig. S12 and S13), display near-perfect discrimination of DH2–DH7. The improved binding specificity exhibited by the double-stranded probes is likely a manifestation of the stringency clamping effect, which is observed with structured probes.45 Thus, formation of recognition complexes with DH2–DH7 would require formation of two energetically destabilized mismatched duplexes, whereas only one mismatched duplex needs to form with single-stranded high-affinity probes like γPNA1.46 Thus, the results highlight the promise of double-stranded probes for specific recognition of mixed-sequence dsDNA target regions.

Conclusion

Chimeric γPNA–Invader probes are strongly energetically activated for recognition of complementary mixed-sequence dsDNA targets since (i) the tethered pyrene moieties of monomer X are poorly accommodated in γPNA–Invader duplexes and forced into extrahelical positions leading to destabilization, and (ii) the individual γPNA and Invader strands that comprise the chimeric probes, display very high cDNA affinity. In addition, the double-stranded nature of the chimeric γPNA–Invader probes precludes potentially recognition-impeding γPNA self-hybridization, which may occur if the dsDNA targets harbor partially self-complementary regions. In this respect, the chimeric γPNA–Invader probes function similarly to pseudo-complementary PNA.11,12 Finally, chimeric γPNA–Invader probes display improved binding specificity relative to single-stranded γPNAs in the studied target contexts, presumably due to stringency clamping effects. In contrast, chimeric LNA–Invader probes are only weakly activated for dsDNA-recognition as the pyrene moieties of monomer X intercalate into and stabilize the probe duplexes.

A comparison of probe types revealed that conventional Invader probes generally recognize the DNA hairpin model targets more efficiently than chimeric γPNA–Invader probes (C50 = 40–50 nM vs. 45–165 nM), whereas single-stranded γPNA – when targeting the self-complementary region studied herein – result in less effective dsDNA-recognition (C50 > 250 nM). Chimeric LNA–Invader and single-stranded LNA probes result in minimal recognition. Thus, chimeric γPNA–Invader probes offer the promise of improving the dsDNA-targeting performance of single-stranded γPNA probes, at least in partially self-complementary target contexts. Hence, additional comparative studies of chimeric γPNA–Invader, single-stranded γPNA, and conventional Invader probes across a wide range of non-self-complementary sequence contexts and target types (e.g., PCR fragments, plasmids, and genomic DNA) – along with continued exploration of these probes for applications in molecular biology, biotechnology, and medicine – are warranted to determine their full scope.

Experimental section

Synthesis and purification of probe strands

The single-stranded Invader, LNA and γPNA strands used herein were originally prepared in connection with a recent study of ours. For details of synthesis, purification, and quality control, see ref. 30.

Thermal denaturation experiments

Concentrations of Invader and LNA strands were estimated using the following extinction coefficients (OD260/μmol): G (12.01), A (15.20), T (8.40), C (7.05), pyrene (22.4).47 Extinction coefficients for γPNA were calculated as previously described.17 Thermal denaturation temperatures (Tms) of duplexes (1.0 μM final concentration of each strand) 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 the thermal denaturation curve (A260vs. T) recorded in medium salt buffer (Tm buffer: 100 mM NaCl, 0.2 mM EDTA, and pH 7.0 adjusted with 10 mM Na2HPO4 and 5 mM Na2HPO4). Strands were mixed in quartz optical cells with a path-length of 1.0 cm and annealed by heating to 85 °C (2 min), followed by cooling to the starting temperature of the experiment. The temperature of the denaturation experiments ranged from at least 15 °C below the Tm to at least 15 °C above the Tm (though not above 95 °C). A temperature ramp of 1.0 °C min−1 was used in all experiments, which resulted in minimal hysteresis (difference in Tm as determined from heating and cooling cycles is <3 °C). Reported Tms are averages of at least two experiments within ±1.0 °C.

Determination of thermodynamic parameters

Thermodynamic parameters associated with duplex formation were determined through baseline fitting of denaturation curves (van't Hoff method) using software provided with the UV-Vis spectrophotometer. Bimolecular reactions, two-state melting behavior, and constant heat capacity are assumed.37 Two denaturation curves per duplex were analyzed at least three times to minimize errors arising from baseline choice.

Optical spectroscopy

Absorption spectra (range 200–600 nm) were recorded at 10 °C using the same samples (i.e., each strand used at 1.0 μM in Tm buffer) and instrumentation as in the thermal denaturation experiments.

Steady-state fluorescence emission spectra were recorded in non-deoxygenated Tm 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 λex = 350 nm and scan speed of 600 nm min−1 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.30 Thus, DNA hairpins (DH) were obtained from commercial sources and used without further purification. Hairpins were digoxigenin (DIG)-labeled using the 2nd generation DIG Gel Shift Kit (Roche Applied Bioscience) 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, γPNA or LNA probes (concentration as specified) were heated (90 °C for 2 min) and 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) at 37 °C for the specified time. In time–course experiments, aliquots were taken at specific time points, flash-frozen in liquid N2, and stored at −20 °C until analysis. Following incubation, loading dye (6×) was added and the mixtures were loaded onto 12% non-denaturing TBE-PAGE gels (45 mM tris-borate, 1 mM EDTA; acrylamide[thin space (1/6-em)]:[thin space (1/6-em)]bisacrylamide (19[thin space (1/6-em)]:[thin space (1/6-em)]1)). Electrophoresis was performed using constant voltage (∼70 V) at ∼4 °C for ∼2 h. Bands were then blotted onto positively charged nylon membranes (∼100 V, 30 min, ∼4 °C) and cross-linked through exposure to UV (254 nm, 5 × 15 W bulbs, 5 min). Membranes were incubated with anti-digoxigenin-alkaline phosphatase Fab 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 dsDNA 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,48 was used to fit following equation to the data: y = C + A(1 − ekt) where C, A and k are constants. The resulting equation was used to calculate C50 values by setting y = 50 and solving for t.

Conflicts of interest

P. J. H. is an inventor on patents pertaining to Invader probes, which have been issued to the University Idaho.

Acknowledgements

We express our gratitude to Saswata Karmakar (Univ. Idaho) for synthesizing 2′-O-(pyren-1-yl)-methyluridine phosphoramidites and to Dale C. Guenther (Univ. Idaho), Prof. Jesper Wengel (Univ. Southern Denmark) and Dr Tumul Srivastava for providing the Invader, LNA and γPNA strands, respectively, used in the present study. This study was supported by awards IF13-001 and IF14-012 from the Higher Education Research Council, Idaho State Board of Education.

Notes and references

  1. M. Duca, P. Vekhoff, K. Oussedik, L. Halby and P. B. Arimondo, Nucleic Acids Res., 2008, 36, 5123–5138 CrossRef CAS PubMed.
  2. K. Kaihatsu, B. A. Janowski and D. R. Corey, Chem. Biol., 2004, 11, 749–758 CrossRef CAS PubMed.
  3. Y. Kawamoto, T. Bando and H. Sugiyama, Bioorg. Med. Chem., 2018, 26, 1393–1411 CrossRef CAS PubMed.
  4. Y. Hari, S. Obika and T. Imanishi, Eur. J. Org. Chem., 2012, 2875–2887 CrossRef CAS.
  5. D. A. Horne and P. B. Dervan, J. Am. Chem. Soc., 1990, 112, 2435–2437 CrossRef CAS.
  6. V. V. Filichev, M. C. Nielsen, N. Bomholt, C. H. Jessen and E. B. Pedersen, Angew. Chem., Int. Ed., 2006, 45, 5311–5315 CrossRef CAS PubMed.
  7. E. M. Zaghloul, A. S. Madsen, P. M. D. Moreno, I. I. Oprea, S. El-Andaloussi, B. Bestas, P. Gupta, E. B. Pedersen, K. E. Lundin, J. Wengel and C. I. E. Smith, Nucleic Acids Res., 2011, 39, 1142–1154 CrossRef CAS PubMed.
  8. K. Kaihatsu, R. H. Shah, X. Zhao and D. R. Corey, Biochemistry, 2003, 42, 13996–14003 CrossRef CAS PubMed.
  9. P. R. Bohländer, T. Vilaivan and H.-A. Wagenknecht, Org. Biomol. Chem., 2015, 13, 9223–9230 RSC.
  10. I. V. Kutyavin, R. L. Rhinehart, E. A. Lukhtanov, V. V. Gorn, R. B. Meyer and H. B. Gamper, Biochemistry, 1996, 35, 11170–11176 CrossRef CAS PubMed.
  11. J. Lohse, O. Dahl and P. E. Nielsen, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 11804–11808 CrossRef CAS.
  12. T. Ishizuka, J. Yoshida, Y. Yamamoto, J. Sumaoka, T. Tedeschi, R. Corradini, S. Sforza and M. Komiyama, Nucleic Acids Res., 2008, 36, 1464–1471 CrossRef CAS PubMed.
  13. M. Hibino, Y. Aiba, Y. Watanabe and O. Shoji, ChemBioChem, 2018, 19, 1601–1604 CrossRef CAS PubMed.
  14. V. V. Filichev, B. Vester, L. H. Hansen and E. B. Pedersen, Nucleic Acids Res., 2005, 33, 7129–7137 CrossRef CAS PubMed.
  15. Y. Aiba, Y. Honda and M. Komiyama, Chem. – Eur. J., 2015, 21, 4021–4026 CrossRef CAS.
  16. D. A. Rusling, V. E. C. Powers, R. T. Ranasinghe, Y. Wang, S. D. Osborne, T. Brown and K. R. Fox, Nucleic Acids Res., 2005, 33, 3025–3032 CrossRef CAS PubMed.
  17. B. Sahu, I. Sacui, S. Rapireddy, K. J. Zanotti, R. Bahal, B. A. Armitage and D. H. Ly, J. Org. Chem., 2011, 76, 5614–5627 CrossRef CAS PubMed.
  18. R. Bahal, B. Sahu, S. Rapireddy, C. M. Lee and D. H. Ly, ChemBioChem, 2012, 13, 56–60 CrossRef CAS PubMed.
  19. S. P. Sau, T. S. Kumar and P. J. Hrdlicka, Org. Biomol. Chem., 2010, 8, 2028–2036 RSC.
  20. S. Rapireddy, R. Bahal and D. H. Ly, Biochemistry, 2011, 50, 3913–3918 CrossRef CAS.
  21. R. Bahal, E. Quijano, N. A. McNeer, Y. Liu, D. C. Bhunia, F. Lopez-Giraldez, R. J. Fields, W. M. Saltzman, D. H. Ly and P. M. Glazer, Curr. Gene Ther., 2014, 14, 331–342 CrossRef CAS PubMed.
  22. A. Orenstein, S. A. Berlyoung, E. E. Rastede, H. H. Pham, E. Fouquerel, T. C. Murphy, J. B. Leibowitz, J. Yu, T. Srivastava, A. B. Armitage and L. P. Opresko, Molecules, 2017, 22, 2117 CrossRef PubMed.
  23. J. Nölling, S. Rapireddy, J. I. Amburg, E. M. Crawford, R. A. Prakash, A. R. Rabson, Y.-W. Tang and A. Singer, mBio, 2016, 7, e00345-00316 CrossRef PubMed.
  24. S. P. Sau, A. S. Madsen, P. Podbevsek, N. K. Andersen, T. S. Kumar, S. Andersen, R. L. Rathje, B. A. Anderson, D. C. Guenther, S. Karmakar, P. Kumar, J. Plavec, J. Wengel and P. J. Hrdlicka, J. Org. Chem., 2013, 78, 9560–9570 CrossRef CAS PubMed.
  25. S. Karmakar, A. S. Madsen, D. C. Guenther, B. C. Gibbons and P. J. Hrdlicka, Org. Biomol. Chem., 2014, 12, 7758–7773 RSC.
  26. D. M. Crothers, Biopolymers, 1968, 6, 575–584 CrossRef CAS PubMed.
  27. H. Ihmels and D. Otto, Top. Curr. Chem., 2005, 258, 161–204 CrossRef CAS.
  28. B. Denn, S. Karmakar, D. C. Guenther and P. J. Hrdlicka, Chem. Commun., 2013, 49, 9851–9853 RSC.
  29. D. C. Guenther, G. H. Anderson, S. Karmakar, B. A. Anderson, B. A. Didion, W. Guo, J. P. Verstegen and P. J. Hrdlicka, Chem. Sci., 2015, 6, 5006–5015 RSC.
  30. R. Emehiser, E. Hall, D. C. Guenther, S. Karmakar and P. J. Hrdlicka, Org. Biomol. Chem., 2020, 18, 56–65 RSC.
  31. H. Asanuma, R. Niwa, M. Akahane, K. Murayama, H. Kashida and Y. Kamiya, Bioorg. Med. Chem., 2016, 24, 4129–4137 CrossRef CAS.
  32. P. Wittung, S. K. Kim, O. Buchardt, P. Nielsen and B. Norden, Nucleic Acids Res., 1994, 22, 5371–5377 CrossRef CAS.
  33. T. Bryld, T. Højland and J. Wengel, Chem. Commun., 2004, 9, 1064–1065 RSC.
  34. V. V. Filichev, U. B. Christensen, E. B. Pedersen, B. R. Babu and J. Wengel, ChemBioChem, 2004, 5, 1673–1679 CrossRef CAS PubMed.
  35. V. Marin, H. F. Hansen, T. Koch and B. A. Armitage, J. Biomol. Struct. Dyn., 2004, 21, 841–850 CrossRef CAS.
  36. H. Kaur, B. R. Babu and S. Maiti, Chem. Rev., 2007, 107, 4672–4697 CrossRef CAS.
  37. J. L. Mergny and L. Lacroix, Oligonucleotides, 2003, 13, 515–537 CrossRef CAS PubMed.
  38. H. Asanuma, T. Fujii, T. Kato and H. Kashida, J. Photochem. Photobiol., C, 2012, 13, 124–135 CrossRef CAS.
  39. K. Kalyanasundaram and J. K. Thomas, J. Am. Chem. Soc., 1977, 99, 2039–2044 CrossRef CAS.
  40. R. R. Avirah and G. B. Schuster, Photochem. Photobiol., 2013, 89, 332–335 CrossRef CAS.
  41. M. Nakamura, Y. Fukunaga, K. Sasa, Y. Ohtoshi, K. Kanaori, H. Hayashi, H. Nakano and K. Yamana, Nucleic Acids Res., 2005, 33, 5887–5895 CrossRef CAS PubMed.
  42. M. E. Østergaard and P. J. Hrdlicka, Chem. Soc. Rev., 2011, 40, 5771–5788 RSC.
  43. Presumably, the second-slowest moving band in lane 2 corresponds to a binary recognition complex between DH1 and γPNA1, akin to the main product formed when DH1 and γPNA1 are incubated (lane 6).
  44. P. J. Hrdlicka and S. Karmakar, Org. Biomol. Chem., 2017, 15, 9760–9774 RSC.
  45. H. Kuhn, D. I. Cherny, V. V. Demidov and M. D. Frank-Kamenetskii, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 7548–7553 CrossRef CAS PubMed.
  46. S. X. Chen, D. Y. Zhang and G. Seelig, Nat. Chem., 2013, 5, 782–789 CrossRef CAS.
  47. N. N. Dioubankova, A. D. Malakhov, D. A. Stetsenko, M. J. Gait, P. E. Volynsky, R. G. Efremov and V. A. Korshun, ChemBioChem, 2003, 4, 841–847 CrossRef CAS PubMed.
  48. A. M. Brown, Comput. Meth. Prog. Biomed., 2001, 65, 191–200 CrossRef CAS.

Footnote

Electronic supplementary information (ESI) available: Representative thermal denaturation curves and electrophoretograms; additional Tm, thermodynamic, dose–response, time–course and binding specificity data; sequences and Tm's for DNA hairpins. See DOI: 10.1039/c9ob02726b

This journal is © The Royal Society of Chemistry 2020