Fluorogenic thiazole orange TOTFO probes stabilise parallel DNA triplexes at pH 7 and above
The instability of DNA triplexes particularly at neutral pH and above severely limits their applications. Here, we demonstrate that the introduction of a thiazole orange (TO) intercalator onto a thymine nucleobase in triplex forming oligonucleotides (TFOs) resolves this problem. The stabilising effects are additive; multiple TO units produce nanomolar duplex binding and triplex stability can surpass that of the underlying duplex. In one example, a TFO containing three TO units increased the triplex melting temperature at pH 7 by a remarkable 50 C relative to the unmodified triplex. Notably, TO intercalation promotes TFO binding to target sequences other than pure polypurine tracts by the use of 5-(1-propynyl)cytosine (pC) against C:G inversions. By overcoming the instability of triplexes across a broad range of pH and sequence contexts, these very simple 'TOTFO' probes could expand triplex applications into many areas including diagnostics and cell imaging.
Introduction<!>Triplex design, synthesis and initial evaluation<!>Stabilisation of triplex inversions<!>Fluorescence melting<!>TOTFO triplex stabilisation at higher pH<!>Evaluation of uorescence enhancement on triplex formation<!>TOTFO triplex studies by other biophysical techniques<!>Conclusions<!>Conflicts of interest
<p>Triplex-forming oligonucleotides (TFOs) bind in the major groove of double stranded DNA (dsDNA) through Hoogsteen or reverse Hoogsteen hydrogen bonding. 1,2 While most methods of sequence-specic DNA recognition rely on denaturation of dsDNA, TFOs do not require this, so they have great potential in areas such as gene inhibition, [3][4][5] DNA repair and recombination studies, 6 inhibition of protein-DNA binding, 7,8 photocrosslinking of duplex DNA, 9 DNA mutagenesis, 10 and nanotechnology. 11 Unfortunately the most stabilising TFOs, those that form parallel triplexes, have much reduced duplex-binding affinity at neutral pH and above owing to the necessity for cytosine protonation. There have been many efforts to overcome the pH-related instability by introducing a variety of sugar, base and phosphate modications into TFOs. [12][13][14][15][16] Furthermore, the requirement for polypurine:polypyrimidine target duplexes is a signicant limitation. In order to overcome this, a range of modied bases have been designed to stabilise pyrimidine:purine C:G and T:A inversions and thereby increase the range of accessible target duplexes. 17,18 Fluorescent TFOs have been used previously. One important application is visualisation of DNA for detection of genomic sequences and selective molecular recognition, [19][20][21] but these applications require a mechanism upon triplex formation for uorescence enhancement, usually through the use of both a uorophore and quencher. In principle, this could instead be achieved via a uorogenic intercalator which would simultaneously increase triplex stability, potentially across a wide pHrange. Indeed intercalators, both chemically attached to the TFO or untethered, have been shown to stabilise triplexes (and other non-canonical DNA structures). [22][23][24][25][26] A suitable design for stabilized uorescent triplexes could be achieved by labelling TFOs with an intercalative uorophore such as TO (Fig. 1A). For many applications, the uorogenic nature of TO is crucial. 27,28 It acts as a "light switch", experiencing a large enhancement in uorescence emission upon intercalation into DNA due to inhibition of rotation of the dye around its methine bridge. In this context, a TO dimer has been incorporated into modied TFOs that are known as 'ECHO probes' (exciton controlled hybridization sensitive uorescent oligonucleotide). 29 However, no triplex formation was seen at pH 8 (essential for important in vitro enzymatic reactions such as PCR), and the stabilising effect was not additive; two TO dimers did not enhance triplex stability over that obtained from a single addition. Other studies have been carried out on parallel triplexes using terminal (3 0 and 5 0 ) or single internal TO units, but in neither case was stable parallel triplexes reported at pH 7. 30,31 In these studies, only a limited range of target sequences was examined.</p><p>Here, we demonstrate that the chemical attachment of a very simple TO B6 modication (1) to the TFOs (TOTFO probes, Fig. 1A) signicantly stabilises parallel triplexes at neutral pH and above with a large increase in uorescence on triplex formation. We also show that this TO modication can be combined with a readily available modied cytosine base, 5-(1propynyl)-dC (pC) to expand triplex target sequences to those containing pyrimidines (C:G inversions in the duplex, Fig. 1C), which are otherwise destabilising. Importantly, multiple additions of TO are shown to extend the duplex binding affinity of the TOTFO into the nanomolar region.</p><!><p>With the aim of increasing the stability of parallel triplexes, a series of modied 15-18mer TFOs were designed to contain 5-(1-propargylamino)-dU (pdU) at various positions for internal attachment of TO. Since TO binds by intercalation, we chose well separated linker positions in the TFO to avoid hindrance of intercalation that might arise from clustered TO units. The triple bond in pdU is known to have a stabilising effect in triplexes due to increased base stacking. 32,33 Other attachments such as 2 0aminoethoxy thymidine and a longer linker at the 5-position of thymine (amino C6-dT) (Fig. 1A) were also investigated, but pdU was ultimately the most effective (Fig. S1 †). Importantly, the triplexes studied were chosen to cover a range of duplex sequence contexts and stacking environments (Fig. 1B). A 'mixed' sequence with 47% G:C duplex content was designed (TRIP-TC) as well as two T-rich sequences (TRIP-T and TRIP-T i ), and two C-rich sequences, (TRIP-C and TRIP-C i ). The subscript 'i' indicates the presence of a C:G inversion in the purine tract of duplex. 5-Methyl-cytosine ( Me C) (M in Fig. 1B) was used at some loci in the TFOs because triplexes containing Me C are slightly more stable compared to those containing cytosine. 34 TO B6 and TO Q6 carboxylic acid active esters were synthesised according to protocol in Section SA.1.1 † and were incorporated into the TFOs by labelling the aminopropargyl groups in the DNA strands using PyBOP coupling reagent (Section SA.2 †). Efficient labelling was conrmed by mass spectrometry (Tables S1 and  S2 †). The melting temperatures (T m values) of the triplexes were then analysed (Table 1 and S3 †). The unlabelled T-rich TFO-T produced a triplex (TRIP-T) with a T m of 43.2 C in 10 mM sodium phosphate/10 mM MgCl 2 buffer at pH 5.8 ('high Mg 2+ buffer') (Entry 1, Table 1). Initially, TO Q6 was attached to the TFO (i.e. through the quinoline moiety) and this provided a large increase (+14.2 C) in triplex stability in high Mg 2+ buffer compared to the unlabelled triplex. However, as found previously for other TO modications, 29 a second TO Q6 unit did not confer any additional triplex stabilisation (Fig. S2 †). When a single TO B6 unit was attached to the TFO, there was a huge increase in T m compared to the unlabelled triplex (+23.3 C) and fortunately, the stabilising effect of TO B6 increased with the addition of a second and third uorophore, reaching 78.0 C (DT m ¼ +34.8 C). Therefore, TO B6 was used for all remaining studies. The melting studies were also carried out in a buffer closer to cellular salt conditions but still at acidic pH and using Na + ions instead of K + ions (low Mg 2+ buffer, 10 mM sodium phosphate/150 mM NaCl/ 2 mM MgCl 2 , pH 5.8). Strong stabilisation was observed with the addition of three TO B6 units giving a remarkable increase in T m of 44.8 C (Table 1, Entries 1-4). Stabilisation was also seen in the TRIP-TC triplex with the addition of three TO units in the TFO, but the overall enhancement in stability (up to 9.7 C) was not as great, presumably because the unlabelled triplex is already very stable at this pH (T m > 70 C), surpassing the stability of the underlying duplex. However, at pH 7 when the unmodied triplex becomes unstable, the increase in T m from TO incorporation is impressive (>20 C) (Table 1, Entries 10 and 11). The effect of sequence on stability of triplexes can be observed in TRIP-C, which is highly unstable but exhibited major stabilisation with the introduction of 3Â TO (+26.2 C in low Mg 2+ buffer) (Table 1, Entries 12-15). Melting curves and additional melting data are in Fig. S3 and Table S3. †</p><p>To further investigate the sequence specicity of TOTFOs, a scrambled duplex sequence was mixed with 1Â TO TFO-T. No triplex formation was observed by UV melting, conrming that the stabilisation gained from intercalation of TO is coupled with retained sequence specicity (Fig. S4 †). To conrm that the TO intercalator in the TFO does not reduce base pair selectivity, we investigated triplexes in which TFO-T (3Â TO) was mixed with a duplex containing an incorrect base pair to give a T.CG, T.TA or T.GC base triplet instead of T.AT. As expected, there is a decrease in triplex stability (À10 to À14 C) when a mismatched triplet is introduced, with triplet stability in the following order: T.AT > T.CG > T.TA > T.GC. This conrms that TO intercalation does not destroy the selectivity of the TFO for its fully complementary duplex (Fig. S5 †).</p><!><p>Having established strong stabilisation of parallel triplexes at uninterrupted polypurine tracts, attention was focussed on triplexes in which the duplex contains a triplex-destabilising base pair inversion. To study this, a C:G base pair was inserted in the poly-A:T or poly-G:C tract of the duplex DNA target (TRIP-T i and TRIP-C i ). Previous studies on modied bases that can recognise and stabilise C:G inversions require multi-step synthesis of special phosphoramidite monomers. 17,35 Here, 5-(1-propynyl)-dC (pC), a commercially available monomer, was used to form a Hoogsteen interaction with the inverted cytosine base of the duplex (a pC.CG triplet). The pC nucleobase has been investigated in TFOs previously for binding to G:C base pairs, where it was destabilising due to its resistance to protonation at N3 (low pK a H). 36 However, for C:G recognition, this is advantageous as protonation of pC is not required (Fig. 1C, S6 †). A comparison to thymine, the most effective natural base to stabilise a C:G inversion (Fig. S5 †), 35 indicated that pC is slightly superior, giving a DT m ¼ +1.2 C and +1.6 C in high and low Mg 2+ buffers respectively (Table 1, Entry 7). To investigate the selectivity of pC for the C:G base pair, TFO-T i was mixed with three different duplexes to form triplexes containing a mismatch triplet (pC.GC, pC.AT and pC.TA instead of pC.CG) (Fig. S7 †). The pC.AT triplet is interesting because the A:T base pair would cause selectivity problems when using T to recognise C:G in a genomic context due to the fact that the T.AT triplet is more stable than T.CG. Fortunately, pC.AT produces a less stable triplex compared to pC.CG at pH 7 (DT m ¼ À12.3 C) and is much less stable than T.AT (DT m ¼ À24.7 C). pC.GC produces a triplex of similar stability to pC.CG in low pH but we see this stability is highly pH-dependent (DT m ¼ À24.6 C) as the pH is increased whilst pC.CG remains far less pH-dependent.</p><p>Incorporation of the C:G inversion into the TRIP-T i and TRIP-C i sequence environments showed that the TRIP-C i triplex is far less stable than TRIP-T i (DT m ¼ À10.8 C, pH 5.8, high Mg 2+ buffer, Table 1, Entries 6 and 17). This is expected as contiguous runs of cytosine bases destabilise triplexes because the pK a H of each individual C-nucleobase is reduced due to their close proximity. 37,38 The inclusion of a single TO unit in TFO-T i led to a large increase in the T m of the TRIP-T i triplex (DT m > 16.0 C) and a remarkable +43.6 C increase in low Mg 2+ buffer for the triplex with three units of TO (Table 1, Entries 7-9). A smaller increase in stability was observed in the TRIP-C i triplex with one TO-addition (DT m $ 3 C, Table 1, Entry 18). However, the T m increased much more (by 16 C) when three TO units were introduced into the TFO-C i (Table 1, Entry 20). Hence, in general TO intercalation and pC as a C:G recognition monomer more than compensates for the destabilising effect caused by the triplet inversion.</p><!><p>As triplexes become more stable, UV monitoring of thermal denaturation is a less reliable determinant of T m because melting of the triplex is obscured by the duplex melting transition (Fig. S8 †) and this can also distort the triplex melting transition. In such cases, uorescence melting can be used instead, because in this technique only the triplex to duplex transition is detectable. 19 Fluorescence melting was therefore employed to analyse triplexes, and in cases where both UV and uorescence melting could be measured the melting temperatures were in good agreement with slight differences due to the DNA concentrations used (1 mM for UV melting, 0.625 mM for uorescence melting). Also, small differences in T m result from the fact that the two techniques monitor different DNA meltingrelated processes. UV melting is sensitive to the dissociation of strands via the unstacking of bases whilst uorescence melting reports on intercalation of the TO into the triplex.</p><!><p>As discussed previously, one of the limitations of triplexes and their applications is the necessity to conduct experiments at low pH. With stabilisation from TO, it is possible to increase the pH and still form stable triplexes. 3Â TO TRIP-T i is one of the most stable triplex at pH 7, with a T m > 70 C (Fig. 2A and B) and the additive effective of TO is clear. Substantial stabilisation is also observed for TRIP-T and TRIP-TC compared to their unlabelled triplexes in low Mg 2+ buffer (Fig. 2C). However, 3Â TO TRIP-C is much less stable (T m $ 35 C). This is because it is rich in cytosine bases (78%) which require protonation to form stable C + .GC triplets and this will not occur at neutral pH. It is noteworthy that we could not observe any triplex formation for unmodied TRIP-C at pH 7 but could overcome this instability with TO attachment. In some sequence contexts, very stable triplexes can be produced at pH 7 with only one TO modication e.g. 1Â TO TRIP-T, T m ¼ 60.0 C and 50.6 C in high and low Mg 2+ buffer respectively (Table 1, Entry 2). Stable triplexes can even be formed above neutral pH; TRIP-TC with 3Â TO has a T m of 54 C at pH 8 (Table S4 †). Hence, these studies demonstrate that triplex formation in biologically compatible conditions is feasible using the simple TO B6 modication.</p><p>The inuence of the T.AT/C + .GC triplet ratio on unlabelled and TO-labelled triplex stability at pH 7 is shown in Fig. 2C. C + .GC-rich triplexes are unstable, T.AT-rich triplexes are much more stable and triplexes with an even mixture of T.AT and C + .GC triplets are the most stable. All benet from the presence of TO immensely (some with DT m > +40 C).</p><!><p>To quantify the enhancement of uorescence intensity upon triplex formation, the uorescence spectra of the TFOs both in the absence and presence of the complementary duplex were measured (Fig. 2D). TFOs excited at 484 nm showed minimal uorescence in single-strand form (ssTFO). Upon addition of the duplex, uorescence intensity was greatly enhanced, 23-fold for the TRIP-T triplex at pH 5.8. At neutral pH, slightly greater enhancement (26-fold) was seen upon triplex formation (Table 2). The uorescent intensity ratios between the free TFOs and the triplexes are given in Table 2. A decrease in the ratio is observed as additional TO units are introduced into the TFO, even though the triplexes with 2 and 3 TO units are much brighter than that with a single TO. This is due to the higher uorescence of the single stranded TFOs that contain multiple TO units. Fluorescence increases slightly at higher pH, and the trends are similar. Further uorescent analysis is provided in Fig. S9. † Quantum yield measurements were carried out on the triplexes (Table S5 †) and lie mostly between 0.13 and 0.23 at pH 7, varying as a function of sequence context. This is in line with published data on thiazole orange in DNA duplexes and higher ordered structures. [39][40][41] For many applications, the inherent uorescence of thiazole orange will be adequate for detection purposes, but in specic cases a brighter and more stable uorophore will be required e.g. for super-resolution imaging of duplex sequences in genomic DNA in live or xed cells. 42 Our TOTFO concept was designed with this in mind; the termini of the TFO are unmodied and therefore available for orthogonal uorescent labelling. This was demonstrated by the synthesis of an ATTO 647N click-labelled TOTFO (TO-ATTO TRIP-T i , Fig. S10 †) which forms a stable triplex with its target duplex.</p><!><p>Triplex formation and stability were also monitored by other techniques. The TRIP-T i triplex was investigated by native polyacrylamide gel-electrophoresis (PAGE) at pH 7 (Fig. 3). Triplexes were separated from the duplex by gel electrophoresis based on charge and size, then visualised by inherent TO uorescence and SYBR Gold staining. The duplex can be identied from lane 1 and the triplex can be observed in lanes 3-7 where the TOTFOs were added to the duplexes. Under SYBR Gold staining it can be seen that the triplex band migrates slightly slower than the duplex band. As expected, decreasing the TFO:duplex ratio results in less intense triplex bands (compare lanes 5, 6, 7). The triplex band was extracted from the gel and analysed by mass spectrometry, which revealed three peaks of the expected masses, conrming the presence of each strand of the triplex (Fig. S11 †).  Fluorescence values are averages of two independent measurements. l ss em ¼ max. emission wavelength of single strand TFO, l T em ¼ max. emission wavelength of formed triplex, F ss @l T em ¼ uorescence intensity of ssTFO at the max. emission wavelength of the triplex, F T @l T em ¼ uorescence intensity of triplex at the max. emission wavelength of the triplex, F T /F ss ¼ ratio of uorescence intensity calculated by comparison of the ssTFO uorescence intensity and the formed triplex uorescence intensity at l T em .</p><p>SPR analysis was carried out to corroborate the gelelectrophoresis and thermal denaturation studies. The unlabelled triplexes TRIP-T i produced a K D of 1.5 mM in accordance with previous reports in which the dissociation constants of triplexes are generally within the micromolar range. 43 A progressive increase in binding was observed with two then three additions of TO, three additions giving a binding constant of 3.9 nM (Fig. S12 †). This data supports the trends observed in the triplex melting studies discussed above.</p><!><p>Triplex stability is greatly enhanced by incorporation of thiazole orange between the 5-position of thymine and the benzothiazole moiety of TO (TO B6 ) in triplex forming oligonucleotides (TOTFOs). Very stable triplexes are obtained at (and above) neutral pH using this straightforward methodology. The stabilising effects of TO are cumulative; the most stable triplexes at pH 7 was formed with three additions of TO in TFOs that target a A:T-rich and mixed-sequence duplexes, and T m increases up to 45 C were achieved. This greatly increased stability has enabled us to expand the range of target duplexes at neutral pH to polypurine sequences that contain a pyrimidine inversion using pC as a C:G recognition monomer. In one such case an increase in T m of 50 C was obtained at pH 7. TOTFOs undergo large enhancement in uorescence upon triplex formation and this could be signicant for many applications. TOTFOs are also substrates for dual dye labelling enabling the attachment of very bright uorophores for stringent biological studies (ATTO-TOTFOs). This simple approach to triplex stabilisation could unlock many practical applications and might also be effective in the stabilisation of highly unstable antiparallel triplexes.</p><p>Finally, the method used in this work for oligonucleotide labelling involved reaction of TO active esters with amino-modied oligonucleotides. Now that we have established which TO-nucleotide is the most effective (TO B6 -pdU) it will be more efficient in future to introduce it directly into TFOs during solidphase synthesis via its phosphoramidite monomer and equip future generations of the probes with substituents that interrogate the target sequence in both the major and minor groove. 44</p><!><p>There are no conicts to declare.</p>