Recognition of mixed-sequence DNA targets using spermine-modified Invader probes

Shiva P. Adhikari , Raymond G. Emehiser , Saswata Karmakar and Patrick J. Hrdlicka *
Department of Chemistry, University of Idaho, 875 Perimeter Drive MS2343, Moscow, ID 83844-2343, USA. E-mail:

Received 31st July 2019 , Accepted 16th August 2019

First published on 19th August 2019

Double-stranded oligodeoxyribonucleotides with +1 interstrand zipper arrangements of 2′-O-(pyren-1-yl)methyl-RNA monomers are additionally activated for highly specific recognition of mixed-sequence DNA targets upon incorporation of non-nucleotidic spermine bulges.

Development of constructs capable of recognizing specific sequences of double-stranded DNA (dsDNA) continues to be an aspirational goal that is fuelled by the promise of tools that will enable site-specific regulation, detection, and manipulation of genomic DNA. Early technologies, such as triplex forming oligonucleotides and peptide nucleic acids (PNAs),1,2 have proven robust but are limited to recognition of extended polypurine targets. Many alternative nucleic acid mimics have been developed3–15 but recognition of mixed-sequence dsDNA sequences at physiologic conditions remains challenging. Even CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) nucleoprotein constructs,16 which have received much recent attention, face many challenges that remain to be resolved, including reducing off-target binding and editing activities, and improving cellular delivery.17

We have previously introduced Invader probes as a potential solution toward specific sequence-unrestricted recognition of dsDNA.18 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).19 This particular monomer arrangement forces two intercalating moieties to compete for the same inter-base-pair region, resulting in localized unwinding and probe destabilization20,21 as the nearest-neighbor exclusion principle22 – which asserts that intercalation is anti-cooperative at adjacent sites23 – is violated. Each individual strand of an Invader probe displays exceptionally high affinity toward complementary DNA (cDNA) as duplex formation results in strongly stabilizing stacking interactions between the intercalators and flanking base-pairs.18–21 The greater stability of the duplexes in the formed recognition complex vis-à-vis the Invader probe and dsDNA target region, provides the driving force for recognition of complementary dsDNA regions, which can be of mixed sequence composition (Fig. 1). Invader probes have, for example, been used to recognize mixed-sequence target regions on Y-chromosomes in fixed nuclei from male bovine kidney cells under otherwise non-denaturing conditions.19

image file: c9ob01686d-f1.tif
Fig. 1 (a) Structures of 2′-O-(pyren-1-yl)methyluridine and spermine monomers used herein. (b) Principle of sequence-unrestricted dsDNA-recognition using bulged Invader probes.

We recently introduced Invader probes that are additionally modified with nonyl (C9) bulges,24 expecting that the non-nucleotidic bulges would promote further denaturation and destabilization of the probe duplex. This, in turn, was expected to increase the availability of the probe's Watson–Crick face for binding and accelerate nucleation with, and invasion of, dsDNA targets. Indeed, we observed that incorporation of C9 bulges at certain positions of Invader probes results in more efficient (>5-fold) and faster (>4-fold) dsDNA-recognition relative to conventional Invader probes at certain conditions.24

Motivated by these findings, we set out to study Invader probes with spermine bulges, which are considerable larger than the C9 bulges (Fig. 1). We were particularly eager to study probes with opposing spermine bulges due to the prospect for additional destabilization and energetic activation for dsDNA-recognition stemming from increased duplex disruption and electrostatic repulsion from the positively charged25 spermine bulges. Towards this end, we synthesized a series of oligodeoxyribonucleotides (ONs) modified with commercially available spermine monomer S25 and 2′-O-(pyren-1-yl)methyluridine monomer X,19 allowing us to evaluate a series of 13-mer spermine-containing Invader and control probes (Table 1 – see ESI for full details). The probe design features two consecutive +1 interstrand zipper arrangements of X monomers (i.e., two energetic hotspots) in the probe center and one or two spermine monomers at one or both termini.

Table 1 Sequences of probes used in this study, Tms of probe duplexes and duplexes between individual probe strands and cDNA, and thermal advantages (TAs) of probesa
    T mTm] (°C)  
ONs Sequence Probe duplex Upper strand vs. cDNA Lower strand vs. cDNA TA (°C)
a ΔTms are calculated relative to the corresponding unmodified dsDNA (Tm = 37.5 °C). Thermal denaturation curves were recorded in medium salt phosphate buffer ([Na+] = 110 mM, [Cl] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4), [EDTA] = 0.2 mM), with each [ON] at 1.0 μM. See main text for the definition of TA. ON7 is so numbered to facilitate comparison with C9-modified Invader probes.24 b Data previously reported in ref. 24.
1[thin space (1/6-em)]:[thin space (1/6-em)]2b image file: c9ob01686d-u1.tif 45.0 [+7.5] 55.5 [+18.0] 55.5 [+18.0] 28.5
3[thin space (1/6-em)]:[thin space (1/6-em)]2 image file: c9ob01686d-u2.tif 38.0 [+0.5] 48.0 [+10.5] 55.5 [+18.0] 28.0
1[thin space (1/6-em)]:[thin space (1/6-em)]4 image file: c9ob01686d-u3.tif 43.0 [+5.5] 55.5 [+18.0] 51.0 [+13.5] 26.0
3[thin space (1/6-em)]:[thin space (1/6-em)]4 image file: c9ob01686d-u4.tif 41.0 [+3.5] 48.0 [+10.5] 51.0 [+13.5] 20.5
7[thin space (1/6-em)]:[thin space (1/6-em)]2 image file: c9ob01686d-u5.tif 23.0 [−14.5] 35.0 [−2.5] 55.5 [+18.0] 30.0
7[thin space (1/6-em)]:[thin space (1/6-em)]4 image file: c9ob01686d-u6.tif 28.0 [−9.5] 35.0 [−2.5] 51.0 [+13.5] 20.5
3c:4c image file: c9ob01686d-u7.tif 30.0 [−7.5] 27.5 [−10.0] 31.5 [−6.0] −8.5
3c:2 image file: c9ob01686d-u8.tif 50.5 [+13.0] 27.5 [−10.0] 55.5 [+18.0] −5.0
1[thin space (1/6-em)]:[thin space (1/6-em)]4c image file: c9ob01686d-u9.tif 52.5 [+15.0] 55.5 [+18.0] 31.5 [−6.0] −3.0

Thermal denaturation temperatures (Tms) were determined for probe duplexes and duplexes between individual probe strands and cDNA (Table 1). As previously reported,24 conventional Invader probe ON1:ON2 is moderately stabilized relative to the corresponding unmodified DNA duplex (ΔTm = 7.5 °C, equivalent to an increase of ∼1.9 °C per modification (mod)), whereas duplexes between individual probe strands and cDNA are extraordinarily stabilized (ΔTm = 18 °C, ΔTm/mod = 9 °C). The resulting thermodynamic driving force for dsDNA-recognition can be assessed by the term thermal advantage given as TA = Tm (upper probe vs. cDNA) + Tm (lower probe vs. cDNA) − Tm (probe duplex) − Tm (dsDNA).19 As expected, conventional probe ON1:ON2 is strongly activated (TA = 28.5 °C, Table 1).

Introduction of a single spermine bulge, as in ON3:ON2 and ON1:ON4, reduces the Tm of the probe duplex by 2–7 °C vis-à-vis the conventional Invader probe, but also decreases the Tms of the corresponding duplexes between individual probe strands and cDNA by an equivalent amount (e.g., compare Tm for ON3 and ON1vs. cDNA). Accordingly, the driving force for dsDNA-recognition remains largely unchanged (TA = 26 and 28 °C for ON1:ON4 and ON3:ON2, respectively). Similar trends were seen with the corresponding C9 bulge-containing Invader probes, except that destabilization was even more pronounced (i.e., incorporation of a C9 unit reduced the Tm by 9–13 °C).24

Invader probe ON7:ON2, in which two spermine monomers are introduced – one monomer near each end of a strand – is very labile (Tm = 23 °C). The stability of the duplex between spermine-modified strand ON7 and cDNA is reduced by a similar amount and consequently ON7:ON2 displays similar dsDNA-recognition potential (TA = 30 °C) as conventional Invader probe ON1:ON2.

Introduction of two spermine bulges opposite of each other does not result in additional probe destabilization relative to the corresponding single bulge probes (compare probe Tms for ON3:ON4, ON3:ON2, and ON1:ON4). This is surprising for at least two reasons: (i) additional electrostatic repulsion between two proximal and positively charged spermine units, akin to the repulsion observed for pseudocomplementary PNA with two opposing lysine units,13 could have been expected, and (ii) Invader probes with two opposing C9 bulges are exceptionally destabilized.24 Since incorporation of the spermine monomers decreases the cDNA-affinity of both probe strands, ON3:ON4 displays less pronounced dsDNA-targeting potential (TA = 20.5 °C) than single bulge probes ON3:ON2 and ON1:ON4 or conventional Invader probe ON1:ON2. Control probe ON3c:ON4c, which lacks the two sequential energetic hotspots, is also less destabilized than expected (compare Tm for ON3c:ON4c, ON3c:cDNA and ON4c:cDNA). Along similar lines, ON7:ON4, which features one isolated and two opposing spermine monomers, denatures less easily (Tm = 28 °C) than ON7:ON2, resulting in reduced dsDNA-recognition potential (TA = 20.5 °C). Collectively, these observations suggest that opposing spermine monomers decrease electrostatic repulsion between probe strands, presumably because their overriding effect is reduction of the net negative charge of the strands rather than mutual interference. Tm measurements performed at low ionic strengths corroborate this conclusion (Table S2). Control experiments entailing Invader probes with small PEG bulges could be carried out to further study the impact of electrostatics vis-à-vis solvation.

As expected, negative controls ON3c:ON4c, ON3c:ON2, and ON1:ON4c are not activated for dsDNA-recognition (TA values <−3.0 °C) as they lack the +1 interstrand zipper arrangements of X monomers that are necessary for probe activation.

An established electrophoretic mobility shift assay, utilizing a digoxigenin (DIG)-labelled DNA hairpin (DH) as a model target,19 was used to evaluate the dsDNA-targeting properties of the probes (Fig. 2a). DH1 is comprised of a 13-mer double-stranded target segment and a T10 loop that covalently links the two stem strands at one end. Recognition of DH1 is expected to result in the formation of a slower-moving ternary recognition complex (RC) upon electrophoretic resolution of incubation mixtures on non-denaturing polyacrylamide gels (Fig. 2b). An initial screen was performed in which DH1 was incubated with a 200-fold molar excess of probes at 25 °C. ON3:ON2 and ON1:ON4 featuring a single spermine bulge, ON7:ON2 with one spermine bulge near each end, and conventional Invader probe ON1:ON2 result in highly efficient recognition of DH1 (Fig. 2b and c). In accordance with the observed TA values (Table 1), all other probes display moderate (ON3:ON4 and ON7:ON4) or minimal recognition of DH1 (ON3c:ON2, ON1:ON4c, and ON3c:ON4c) (see also Table S3). A subsequent screen in which a 100-fold molar probe excess was incubated with DH1 at 8 °C (Fig. S11), revealed, remarkably, that dsDNA-recognition also is possible at low experimental temperatures, albeit being less efficient. Spermine-containing Invader probes ON3:ON2, ON1:ON4, and ON7:ON2 result in similar to slightly improved recognition of DH1vis-à-vis conventional Invader probe ON1:ON2 (23–34% vs. 20%, respectively), whereas all other probes displayed minimal or no recognition (Fig. 2c and Table S3).

image file: c9ob01686d-f2.tif
Fig. 2 (a) Illustration of the electrophoretic mobility shift assay used to evaluate dsDNA-recognition of bulged Invader probes. (b) Representative gel electrophoretograms for recognition of model dsDNA target DH1 (34.4 nM) by various probes (6.88 μM; 25 °C). (c) Histogram depicting the average outcome of at least three recognition experiments at 8 °C or 25 °C using 100- or 200-fold probe excess, respectively (see Table S3 for tabulated data); error bars represent standard deviation. DIG-labelled DH1 (5′-GGTATATATAGGC-T10-GCCTATATATACC-3′) (Tm = 58.5 °C) was incubated with pre-annealed probes in HEPES buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, pH 7.2, 10% sucrose, 1.44 mM spermine tetrahydrochloride) for 17 h.

Based on these preliminary results, Invader probes ON3:ON2 and ON7:ON2, with one and two spermine bulges, respectively, were selected for further evaluation and comparison with conventional Invader probe ON1:ON2. Dose–response profiles were determined for these probes at 8 °C, 25 °C, and 37 °C (Fig. 3 and S12). At 8 °C, ON3:ON2 and ON7:ON2 result in more efficient recognition than ON1:ON2 as indicated by lower C50 values, i.e., probe concentrations resulting in 50% recognition of DH1 (∼2.8 μM, ∼1.8 μM, and ∼5.6 μM, respectively, Table 2). Given the similar dsDNA-targeting driving force of these probes, these trends are most likely a result of the lower Tms and more readily denaturing nature of the bulge-containing probes. Note, the probe–target duplexes formed as part of the recognition process are expected to be fully stable at this temperature (Tms > 35 °C). Also, denaturation of DH1 is unlikely at 8 °C (Tm = 58.5 °C, Table S4), which strongly suggests that dsDNA-recognition proceeds via a double-duplex invasion mechanism (where partial melting of DH1 and Invader probes reveals nucleation sites serving as initiation sites for formation of probe–target duplexes) rather than a strand exchange mechanism (which would entail complete denaturation of DH1 and Invader probes, followed by reassembly of the most thermodynamically favorable complexes).

image file: c9ob01686d-f3.tif
Fig. 3 Dose–response curves for recognition of DH1 by Invader probes ON1:ON2, ON3:ON2 and ON7:ON2 at (a) 8 °C, (b) 25 °C, and (c) 37 °C. Experimental conditions are outlined in Fig. 2. Bars denote standard deviation (three independent experiments).
Table 2 C 50 values for recognition of DH1 by select Invader probesa
Probe C 50, 8 °C (μM) C 50, 25 °C (μM) C 50, 37 °C (μM)
a Calculated from dose–response curves shown in Fig. 3.
ON1:ON2 5.6 0.5 0.7
ON3:ON2 2.8 0.5 1.2
ON7:ON2 1.8 1.2 1.8

Recognition of DH1 is more efficient at 25 °C (C50 values: 0.5 μM, 0.5 μM, and 1.2 μM, for ON1:ON2, ON3:ON2, and ON7:ON2, respectively, Table 2), presumably because localized probe denaturation is more pronounced at this experimental temperature (DH1 is still expected to be fully hybridized). Consistent with this, the improvement is most pronounced for the highest-melting probe ON1:ON2 (C50 values of 5.6 μM and 0.5 μM at 8 °C and 25 °C, respectively), and least pronounced for the lowest-melting probe ON7:ON2 (C50 values of 1.8 μM and 1.2 μM at 8 °C and 25 °C, respectively).

Recognition of DH1 is less efficient at 37 °C (Table 2) presumably as the higher experimental temperature impacts the stability of the ternary recognition complexes in this particular sequence context. The trend is more pronounced for bulge-containing Invader probes, which display lower cDNA affinity than conventional Invader probe ON1:ON2 (compare Tms for ON3:cDNA, ON7:cDNA, and ON1:cDNA, Table 1).

Finally, the binding specificity of these three Invader probes was evaluated. Probes were incubated with DNA hairpins DH2–DH7, which have fully base-paired double-stranded stems that differ in sequence relative to the probes at either the 6- or 9-position (Fig. 4; for sequences and Tms of DH1–DH7 see Table S4). Remarkably, ON3:ON2 and ON7:ON2, as well as, reference probe ON1:ON2 display perfect discrimination of the singly mismatched dsDNA-targets at conditions that result in complete recognition of complementary target DH1 (100-fold probe excess, 25 °C, 17 h, Fig. 4). The double-stranded Invader probes likely display superb discrimination of mismatched dsDNA targets due to stringency clamping effects26 and because binding to DH2–DH7 would require the formation of recognition complexes with two energetically unfavourable mismatched duplexes.27

image file: c9ob01686d-f4.tif
Fig. 4 Binding specificity of spermine bulge-containing Invader probes. Top panel: Illustration of non-complementary targets DH2DH7 (see Table S4 for sequences). Other panels: Representative electrophoretograms from experiments in which a 100-fold excess of ON7:ON2, ON3:ON2, and ON1:ON2 were incubated with DH1–DH7 (34.4 nM) at 25 °C for 17 h. Experimental conditions are as outlined in Fig. 2.

In summary, Invader probes with one or two spermine bulges denature more readily than conventional Invader probes and result in improved and very specific recognition of mixed-sequence dsDNA targets at low incubation temperatures. Invader probes that are additionally activated through incorporation of spermine and other non-nucleotidic bulges are expected to be particularly useful for recognition of high-melting DNA targets, i.e., extended and/or highly GC-rich regions, which currently represent challenging targets as the corresponding conventional Invader probes are very high-melting. Studies along these lines are ongoing.

Conflicts of interest

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


Initial contributions by Brooke A. Anderson (Univ. Idaho) are appreciated. 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, C. Nielsen, C. H. Bomholt 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. R. Bahal, B. Sahu, S. Rapireddy, C.-M. Lee and D. H. Ly, ChemBioChem, 2012, 13, 56–60 CrossRef CAS PubMed.
  10. P. R. Bohlander, T. Vilaivan and H. A. Wagenknecht, Org. Biomol. Chem., 2015, 13, 9223–9230 RSC.
  11. I. V. Kutyavin, R. L. Rhinehart, E. A. Lukhtanov, V. V. Gorn, R. B. Meyer Jr. and H. B. Gamper Jr., Biochemistry, 1996, 35, 11170–11176 CrossRef CAS PubMed.
  12. J. Lohse, O. Dahl and P. E. Nielsen, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 11804–11808 CrossRef CAS PubMed.
  13. 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.
  14. M. Hibino, Y. Aiba, Y. Watanabe and O. Shoji, ChemBioChem, 2018, 19, 1601–1604 CrossRef CAS PubMed.
  15. V. V. Filichev, B. Vester, L. H. Hansen and E. B. Pedersen, Nucleic Acids Res., 2005, 33, 7129–7137 CrossRef CAS PubMed.
  16. P. D. Hsu, E. S. Lander and F. Zhang, Cell, 2014, 157, 1262–1278 CrossRef CAS PubMed.
  17. A. C. Komor, A. H. Badran and D. R. Liu, Cell, 2017, 168, 20–36 CrossRef CAS PubMed.
  18. P. J. Hrdlicka, T. S. Kumar and J. Wengel, Chem. Commun., 2005, 4279–4281 RSC.
  19. 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.
  20. 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.
  21. S. Karmakar, A. S. Madsen, D. C. Guenther, B. C. Gibbons and P. J. Hrdlicka, Org. Biomol. Chem., 2014, 12, 7758–7773 RSC.
  22. D. M. Crothers, Biopolymers, 1968, 6, 575–584 CrossRef CAS PubMed.
  23. C. Tsai, S. Jain and H. M. Sobell, J. Mol. Biol., 1977, 114, 301–315 CrossRef CAS PubMed.
  24. D. C. Guenther, S. Karmakar and P. J. Hrdlicka, Chem. Commun., 2015, 51, 15051–15054 RSC.
  25. R. Noir, M. Kotera, B. Pons, J.-S. Remy and J.-P. Behr, J. Am. Chem. Soc., 2008, 130, 13500–13505 CrossRef CAS PubMed.
  26. V. V. Demidov and M. D. Frank-Kamenetskii, Trends Biochem. Sci., 2004, 29, 62–71 CrossRef CAS PubMed.
  27. S. X. Chen, D. Y. Zhang and G. Seelig, Nat. Chem., 2013, 5, 782–789 CrossRef CAS PubMed.


Electronic supplementary information (ESI) available: Experimental protocols; HPLC and MS data for ONs; representative thermal denaturation curves and electrophoretograms; additional Tm data; sequences and Tm values of DNA hairpins. See DOI: 10.1039/c9ob01686d

This journal is © The Royal Society of Chemistry 2019