Alternate dab-aegPNAs: synthesis, nucleic acid binding studies and biological activity

Abstract

As part of our research on new oligonucleotide analogs for therapeutic and diagnostic use, here we explored the ability of an alternate dab-aegPNA oligomer to bind complementary natural nucleic acids. The alternate homothymine dab-aegPNA, synthesized following a chirally safe procedure and fully characterized by ESIMS and CD, was capable of forming hybrids with complementary DNA and RNA with enhanced thermal stability in comparison to natural oligomers, as shown by CD and UV spectroscopies. The stoichiometry of the complexes formed was determined by CD titration experiments that suggested triple helices formation. With respect to an analogous t₁₂ strand composed entirely of aegPNA, the chiral alternate t₁₂ oligomer presented an enhanced solubility in aqueous medium and did not form aggregates. Human serum stability assays performed on the new alternate oligomer evidenced a noteworthy enzymatic resistance. Moreover, the efficiency of dab-aegPNA in interfering with the reverse transcription of eukaryotic mRNA, and the absence of cytotoxic effects of the new analog were demonstrated, encouraging us to further study this chiral PNA analog in view of its possible in vivo/in vitro biotechnological applications.

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Introduction

The development of synthetic oligodeoxyribonucleotide (ODN) analogs able to bind natural nucleic acids in a sequence-specific manner has received a remarkable scientific interest for decades due to their biomedical and bioengineering relevance, leading to a number of artificial molecules containing different modifications in the backbone and/or in nucleobases respect to natural oligonucleotides.^1–3 More particularly, aegPNA (aminoethylglycylPNA), introduced for the first time in 1991 by Nielsen et al.⁴ and containing an artificial pseudo-peptide chain in place of the sugar-phosphate backbone (Fig. 1), is one of the most interesting ODN mimics being, able to form very stable complexes with DNA and RNA characterized by high sequence-specificity.⁵ However, some drawbacks, like (1) poor water solubility due to their charge-neutral nature, (2) self-aggregation to a degree dependent on the length of the oligomer and purine–pyrimidine ratio, (3) inefficient cell uptake, limit aegPNA applications within biotechnology .⁵

Fig. 1 Comparison of aeg-, amp- and dabPNA monomeric units.

Moreover, an important characteristic for achieving high ODN-binding specificity resides in the ability of an ODN mimic to discriminate between parallel and antiparallel orientations during hybridization. Interestingly, the introduction of stereogenic centres in PNA structure allows for an improved binding mode discrimination, as well-described in several literature examples reporting PNA analogs with entirely or partially chiral polyamide backbones. In most cases chiral centres are provided by means of amino acid units at the aegPNA termini (especially lysines and arginines) or directly into the PNA backbone (δ-ornPNA, aelPNA, ampPNA, γ-dabPNA, ε-lysPNA, ε-lysPNA/γ-dabPNA, etc.; where orn stand for ornithyl-, ael for aminoethyllysyl-, amp for 4-aminoprolyl-, dab for diaminobutyryl-, and lys for lysyl-).^6–15

Among the chiral PNA analogues, ampPNA could be considered as derived from the aegPNA by the introduction of a methylene bridge between β-carbon of the aminoethyl moiety and the α-carbon atom of the glycine unit (Fig. 1). Inclusion of a single 4-aminoproline at the N-terminus or in the interior of an aegPNA sequence led to oligomers still able to bind native complementary nucleic acids, while fully amp-modified sequences did not bind natural targets suggesting that in homo-oligomer, inter-nucleoside distances are too low. Furthermore, alternate amp-aegPNAs demonstrated higher binding affinities towards natural nucleic acids than pure aegPNA.¹⁶

Recently, as part of our research on new ODN-analogs for biotechnological applications, we reported the enantioselective synthesis of dabPNA monomers and their optically pure oligomerization to nucleo-γ-peptides.^10,11 Our interest in chiral PNA containing DABA (diaminobutyric acid) arises from (1) the possibility to discriminate between parallel/antiparallel orientations during nucleic acid hybridization; (2) the biological activity and enzymatic resistance of γ-aminoacid-containing compounds,^17,18 (3) the natural occurrence of DABA in vegetal tissue of several plants,^18,19 (4) the recent importance in prebiotic life attributed to diaminoacids after their recovery in stellar soil.^20,21 Interestingly, dabPNA can be regarded as the acyclic form of ampPNA (Fig. 1) with only one chiral centre, and differs from aegPNA for a one atom shorter backbone and a one atom longer backbone-nucleobase linker. We previously found that homo-oligomers made of dabPNA units are not able to bind natural nucleic acids, in analogy to ampPNA.^10,11 Nevertheless, we also demonstrated the formation of stable complexes between dabPNA and complementary aegPNA.¹² Since it is known from literature that alternate amp-aegPNA can bind complementary DNA , we intended to ascertain if also an alternate dab-aegPNA oligomer was able to form complexes with DNA and RNA. Here, we report the synthesis, chemical-physical characterization and enzymatic resistance of a novel alternate dab-aegPNA oligomer. Hybridization studies with complementary DNA and RNA were performed by CD‡ and UV spectroscopies. In addition, biological activity and cytotoxicity assays , performed on the new analog in order to evaluate its potential biomedical applications, revealed interesting results discussed below.

Results and discussion

Fmoc-protected L-dabPNA thymine monomer (1, Fig. 2) was obtained by a two-steps procedure, already described in our previous work.^10,11HPLCpurification of 1 was preceded by a precipitation step in order to remove the major part of activator by-products and thus reduce the overall processing time.

Fig. 2 Alternate dab-aegPNA homothymine dodecamer (2) and the relative building blocks.

Oligomer 2 was synthesized manually using solid phase techniques by the alternating use of commercial Fmoc-protected aegPNA and L-dabPNA thymine monomers, ultimately obtaining a dodecamer (Fig. 2). AegPNA monomers were coupled on solid support following standard synthetic protocols, while dabPNA monomers were introduced by a procedure, already employed for the synthesis of homo-L-dabPNAs,^10–12 that ensured retention of optical purity during the coupling steps (Supplementary Scheme S1 in the ESI† ). The terminal amino group was capped by acetylation to prevent side reactions of the detached oligomer in neutral/basic medium caused by the free NH₂. After cleavage from the solid support, precipitation from cold diethyl ether and purification by RP-HPLC, the homothymine t₁₂ strand was obtained in 4.5% yield.

Characterization of oligomer 2 was performed by LC-ESIMS which confirmed the identity of the product (Supplementary Fig. S1 in the ESI† ).

Subsequently, the structural properties of the dab-aegPNA 2 as well as its ability to form complexes with complementary natural nucleic acids were investigated by CD and UV spectroscopies.

Firstly, the CD spectrum of the single strand in 10 mM phosphate buffer (pH 7.5) was recorded in order to evaluate a possible pre-organization of the homothymine alternate molecule. With respect to the achiral aegPNAs that do not show any CD signal, the chiral alternate oligomer 2 presented weak CD signals with a negative band centred around 270 nm at room temperature, in analogy to the CD behaviour already reported for other nucleopeptides, like δ-ornPNA,⁶ γ-dabPNA^10–13 or ε-lysPNA,^14,15 containing L stereocentres. The variation of the CD profile of 2 with the temperature confirmed a certain degree of structural pre-organization for the dab-aegPNA 2 in aqueous medium (Fig. 3).

Fig. 3 CD spectra of dab-aegPNA 2 at various temperatures (6.4 μM in 10 mM phosphate buffer, pH 7.5).

Regarding the binding with DNA and RNA, CD binding experiments were performed in a tandem cell recording the “sum” CD spectra of the separated components and the “mix” CD, recorded after cell mixing, relative to an excess of alternate compound 2 with alternatively dA₁₂ and polyA. The difference observed between the “sum” and “mix” CD spectra revealed the formation of complexes in both cases (Fig. 4).

Fig. 4 Overlapped SUM (dashed line) and MIX (solid line) CD spectra of the alternate dab-aegPNA t₁₂ with dA₁₂ (A) and PolyA (B) (tandem cell).

CD titrations were performed in order to determine the binding stoichiometry for the dab-aegPNA/dA₁₂ and dab-aegPNA/PolyA complexes. The CD variation was followed by adding increasing amounts of alternate PNA2 to dA₁₂ and PolyA in 10 mM phosphate buffer (pH 7.5). Adding more than 2 equivalents of alternate dab-aegPNA, no substantial CD variation was revealed, according to a 2 : 1 stoichiometry in both cases required for (PNA)₂/DNA and (PNA)₂/RNA triple helices formation (Fig. 5).

Fig. 5 CD titration of 4 μM dA₁₂ (A) and 10 μM PolyA (B) with the alternate dab-aegPNA t₁₂ (10 mM phosphate buffer, pH 7.5). Black (only DNA or RNA), red (0.5 eq. t₁₂), blue (1 eq.), magenta (1.5 eq.), green (1.75), grey (2 eq.), purple (2.5 eq.). CD variation at 262 nm are reported in the insets.

Subsequently, we studied the thermal stability of dab-aegPNA/DNA and dab-aegPNA/ RNA complexes by UV spectroscopy. The sigmoidal shapes of UV thermal denaturation curves (Fig. 6a) confirmed the formation of 2 : 1 (in bases) complexes 2/dA₁₂ and 2/PolyA, and showed a T_m of 23.1 °C and 27.0 °C, respectively (Fig. 6b). The processes were reversible and the pairings were completed in about 22 °C (44 min at 0.5 °C min⁻¹) for DNA, and 27 °C (54 min at 0.5 °C min⁻¹) for PolyA complexes.

Fig. 6 (a) UV melting curves in 10 mM phosphate buffer (pH 7.5) of the complexes: dA₁₂/dT₁₂ (dashed line); dA₁₂/dab-aegPNA t₁₂ (1 : 2, solid line); PolyA/dab-aegPNA t₁₂ (1 : 2 in bases, pointed line); (b) T_m of the complexes.

In the hypothesis of a triplex formation between homothymine dab-aegPNA and complementary DNA or RNA, the presence of only one phase transition in the UV melting curves (Fig. 6a) would suggest that no duplex intermediate was effectively formed, in agreement with the reported behaviour of other PNA triplexes.²²

Interestingly, the T_m of the dA₁₂/dab-aegPNA t₁₂ complex resulted about 7 degrees higher than that of the natural dA₁₂/dT₁₂ complex (Fig. 6b), even if it was lower with respect to the complex formed between a dA₁₂ and a t₁₂ made entirely of aegPNA, which showed a T_m > 70 °C (data not shown). Nevertheless, in comparison to a full-aegPNA t₁₂ strand, the chiral alternate t₁₂ oligomer presented an enhanced solubility in aqueous medium and did not form aggregates.

The enzymatic resistance of the new alternate nucleopeptide was investigated by incubating oligomer 2 in fresh human serum at 37 °C and analyzing, by RP-HPLC, samples withdrawn from the reaction mixture at various times (Fig. 7). In analogy to aegPNA, also the alternate nucleopeptide was very stable to enzymatic degradation, being still present even after 24 h, despite natural dT₁₂ that completely disappeared after 1 h (data not shown).

Fig. 7 RP-HPLC analysis of the serum stability assay on dab-aegPNA t₁₂ (2).

Furthermore, we tested the effects of nucleopeptide 2 on the viability of cultured cells. HeLa cells were incubated for 48 h at 37 °C in the presence of 0.1, 1, 10, 25 and 50 μM alternate dab-aegPNA t₁₂. Cell viability was assessed by the MTT reduction assay : no apparent cytotoxic effect was observed.

The biological activity of the alternate nucleopeptide was determined by interference of oligomer 2 with reverse-transcription (RT) reaction using total RNA from HeLa cells. In this experiment RT was performed using dT₁₅ primer and alternatively various amounts of oligomer 2 (0, 2.5 and 25 μg) and 2.5 μg of aegPNA t₁₂ as a positive control. Then, cDNAs obtained were amplified by PCR using gene-specific primers for GAPDH and β-actin (constitutively expressed genes). We found that in the presence of the new alternate nucleopeptide 2 the amplification of both genes was reduced. In particular, the reduction was quantified as 75% and 99% for GAPDHgene (lanes 4 and 5, Fig. 8) and 75% and 96% for β-actin gene (lanes 7 and 8, Fig. 8) when compared with PCR amplification in absence of oligomer 2 (lanes 3 and 6, Fig. 8). Total suppression of gene amplification was obtained with 0.25 μg of aegPNA t₁₂ (lane 2, Fig. 8). When oligomer 2 was used as primer in RT reaction in absence of dT₁₅ primer no PCR amplification products were detected (data not shown).

Fig. 8 Agarose gel electrophoresis analysis of RT-PCR products. RT-PCR amplification of GAPDH (lanes 2–5) and of β-actin (lanes 6–8) genes. Lane 1: molecular markers (M.M.); Lane 2: 0.25 μg of aegPNA t₁₂ (positive control); Lane 3–5: 0, 2.5 and 25 μg of dab-aegPNA 2; Lane 6–8: 0, 2.5 and 25 μg of dab-aegPNA 2.

We suggest for our analog a mechanism of inhibition of the reverse transcription similar to that of standard aegPNAs, which inhibit the action of the Reverse Transcriptase by competing with the primer for binding to the template strand, without any direct PNA-enzyme interaction.²³

Experimental

Reagents and solvents

HATU, was purchased from Novabiochem. Boc-L-DABA(Fmoc)-OH was from Bachem. Anhydroscan DMF and NMP were from LabScan. Piperidine was from Biosolve. Solvents for HPLCchromatography and acetic anhydride were from Reidel-de Haën. PolyA, TFA, TMP, Rink-amide resin were Fluka. TFA (for HPLC) was from Romil. Diethyl ether was from Carlo Erba.

Apparatus

Centrifugations were performed on a Z 200 A Hermle centrifuge. Products were analysed and characterized by LC-MS on an MSQ mass spectrometer (ThermoElectron, Milan, Italy) equipped with an ESI source operating at 3 kV needle voltage and 320 °C, and with a complete Surveyor HPLC system, comprising an MS pump, an autosampler, and a PDAdetector, by using a Phenomenex Jupiter C18 300 Å (5 μm, 4.6 × 150 mm) column. Gradient elution was performed by using increasing amounts of acetonitrile (0.05% TFA) in water (0.05% TFA), monitoring at 260 nm, with a flow rate of 0.8 ml min⁻¹. Semi-preparative purifications were performed on a Hewlett Packard/Agilent 1100 series HPLC, equipped with a diode array detector, by using a Phenomenex Juppiter C18 300 Å (10 μm, 10 × 250 mm) column. Gradient elution was performed at 45 °C (monitoring at 260 nm) by building up a gradient starting with buffer A (0.1% TFA in water) and applying buffer B (0.1% TFA in acetonitrile) with a flow rate of 4 ml min⁻¹. Samples were lyophilized in a FD4 Freeze Dryer (Heto Lab Equipment) for 16 h. CD spectra were obtained on a Jasco J-810 spectropolarimeter. UV spectra and UV melting experiments were recorded on a UV-Vis Jasco model V-550 spectrophotometer equipped with a Peltier ETC-505T temperature controller. UV and CD measurements were performed in Hellma quartz Suprasil cells, with a light path of 1 cm and 2 × 0.4375 cm (Tandem cell).

Solid phase synthesis of oligomer 2

Thymine aegPNA monomer was purchased from Link technologies while thymine dabPNA monomer (1, Fig. 2) was realized starting from the commercial Boc-L-DABA(Fmoc)-OH following the synthetic procedure that we previously reported.^10,11 Solid phase synthesis was carried out in short PP columns (4 ml) equipped with a PTFE filter, a stopcock and a cap on a Rink-amide resin using the peptide-like Fmoc chemistry. Ac-(t_aeg–t_dab)₆–NH₂ (2, Fig. 2) was assembled on Rink-amide-NH₂ resin (0.5 mmol g⁻¹, 16 mg, 8 μmol) using the standard PNA protocol for the insertion of the aegPNA units and following an optimized procedure for dabPNA monomers introduction (Supplementary Scheme S1 in the ESI† ). More particularly, a mixture of Fmoc-L-t_dabPNA-OH (0.2 M in NMP, 120 μl, 24 μmol, 3 eq), HATU (0.18 M in DMF, 120 μl, 21.6 μmol, 2.7 eq) and 200 μl of DMF was introduced into the reactor. Subsequently, 200 μl (40 μmol, 5 eq.) of a TMP solution (0.2 M in NMP) was added to the stirred reaction in 4 portions over 1 h. Capping was performed with 5% (Ac)₂O/6% lutidine for 5 min, while Fmoc deprotection of dabPNA amino group was obtained with 20% piperidine in DMF for 8 min. Couplings of L-dabPNA and aegPNA monomers were checked by measuring the absorbance of the released Fmoc group (ε₃₀₁ = 7800) after treatment with piperidine solution (UV Fmoc test) in order to evaluate the incorporation yields of each monomer. The overall yield of 2 calculated on the basis of the UV Fmoc test was 9%. Oligomer 2 was cleaved from the solid support under acidic conditions (TFA/m-cresol 4 : 1, v/v, 1 ml) and recovered by precipitation with cold diethyl ether, centrifugation and lyophilization. The dodecamer 2 was purified by semipreparative HPLC using a linear gradient of 5% (for 5 min) to 25% B in A over 25 min: t_R = 24.0 min; UV quantification of the purified product gave 360 nmol of 2; ESI-MS (Supplementary Fig. S1 in the ESI† ) m/z: 1628.67 (found), 1628.09 (expected for [C₁₃₄H₁₇₃N₄₉O₄₉ + 2H]²⁺); 1085.38 (found), 1085.74 (expected for [C₁₃₄H₁₇₃N₄₉O₄₉ + 3H]³⁺).

UV and CD studies

Purified oligomers were dissolved in a known amount of milliQ water and quantified by UV measurements (T = 85 °C, absorbance value at λ = 260 nm). The epsilon values used for the quantification of the oligomer 2 (103.2 mM⁻¹) were calculated using the molar extinction coefficient of thymine aegPNA monomer (8.6 mM⁻¹).

Thermal melting curves were obtained by recording the UV absorbance at 260 nm by increasing the temperature at a rate of 0.5 °C min⁻¹. T_m values were calculated by the first derivative plot. CD spectra were recorded from 320 to 200 nm: scan speed 50 nm min⁻¹, data pitch 2 nm, band width 2 nm, response 4 sec, 5 accumulations.

Serum stability assays

1.6 μl of oligomer 2 (0.94 mM in phosphate buffer, pH 7.5) was added to 98.4 μl of 100% fresh human serum in a micro-vial and the mixture was incubated at 37 °C. Aliquots (10 μl) were taken at 0, 1, 2, 24 hours, quenched by adding 10 μl of 7 M urea solution, kept at 95 °C for 2 min and then stored at −20 °C until subsequent analysis. The withdrawn samples were analyzed by HPLC on a Phenomenex Juppiter C18 300 Å (5 μm, 4.6 × 250 mm) column using a linear gradient of 8% (for 5 min) to 40% B in A over 30 min.

Cell culture

HeLa cells (ATCC, USA) were grown in RPMI1640 medium supplemented with 10% FBS, 1% glutamine, 100 U ml⁻¹penicillin and 100 μg ml⁻¹streptomycin (Invitrogen, Carlsbad, CA) at 37 °C in humidified air with 5% CO₂.

Cytotoxicity assay

Cells were plated in 96-well plates (100 μl per well) at a density of 2.5 × 10³ cells per well. Various amount of oligomer 2 was added to the cells 24 h after seeding obtaining the following final concentrations: 0.1, 1, 10, 25 and 50 μM. After 48 h incubation cell viability was determined by the MTT (Sigma-Aldrich) assay . MTT reagent, dissolved in DMEM in the absence of phenol red, was added to the cells (100 μl per well, 0.5 mg ml⁻¹ final concentration). After 4 h at 37 °C, the culture medium was removed and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 N HCl (100 μl/well). Absorbance values of blue formazan were determined at 595 nm using an automatic plate reader (Biorad).

RT-PCR

Total RNA was extracted from the cellular lysate by using Tri-reagent^TM (Sigma Aldrich, S. Luis, MO) following the manufacturer’s instructions. RT was performed using 0.5 μg of total RNA, 200 U of MMLV Reverse TranscriptaseRNase H- (Finnzymes, Espoo, Finland), dNTPs, 250 ng of dT₁₅ primer (Roche, Switzerland) and alternatively various amounts of oligomer 2 (0, 2.5 and 25 μg) and 2.5 μg of aegPNA t₁₂. The mixtures containing RNA and homothymine oligomers were preincubated for 10 min. Reaction temperature was set at 22 °C for 1 h. After RT, PCR assays were carried out for monitoring human β-actin and GAPDHtranscripts using the following primers (purchased by Sigma-Genosys Ltd): forward primer TGAGACCTTCAACACCCC, reverse primer CAGGAAGGAAGGCTGGAA; forward primer ATGGGGAAGGTGAAGGTC, reverse primer 5′-GTCATGGATGACCTTGGC-3′, respectively. The PCR program was as follows: [(95 °C, 5 min) × 1 cycle, (95 °C, 1 min; 58 °C, 1 min; 72 °C, 1 min) × 25 cycles]. PCR products were analyzed on 1% agarose gel with 1 × TAE buffer and visualized by ethidium bromide staining. Gel images were captured by a ChemiDoc™ XRS system and analyzed by Quantity-One software (Biorad, Hercules, CA).

Conclusions

In the present work we report the synthesis of a novel nucleopeptide containing alternate achiral aegPNA and chiral dabPNA repeating units.

By a synthetic point of view the oligomerization was performed in solid phase combining the standard protocol used for aegPNA with a chirally safe procedure for the coupling of dabPNA. In this way, we were able to synthesize the t₁₂ oligomer 2 that was purified by RP-HPLC and characterised by LC-ESIMS.

The ability of such hetero-oligomer to bind complementary DNA and RNA molecules was firstly verified by CD experiments that evidenced the formation of complexes involving oligomer 2 and dA₁₂ or PolyA, suggesting the adoption of triple helix structures. Moreover, the sigmoidal shapes of UV melting curves confirmed the formation of 2/dA₁₂ and 2/PolyA complexes showing T_m of 23.1 °C and 27.0 °C, respectively. Interestingly, the T_m of the dab-aegPNA t₁₂/dA₁₂ complex resulted about 7 degrees higher than that of the natural dT₁₂/dA₁₂ complex, even if it was lower with respect to the complex formed between a dA₁₂ and a t₁₂ made entirely from aegPNA. Nevertheless, in comparison to aegPNA the chiral analog presented an enhanced solubility in aqueous medium and did not form aggregates.

The biological activity of the alternate t₁₂ nucleopeptide was evaluated estimating the interference of oligomer 2 on the reverse-transcription reaction, taking advantage of the presence of polyadenylic tail of eukaryotic mRNA. We found that in presence of 2 the amplification of GAPDH and β-actin genes was reduced, confirming that alternate nucleopeptide binds polyA tails displacing dT₁₅ primer by a strand invasion mechanism. All these findings, jointly with the remarkable enzymatic stability and the absence of cytotoxicity demonstrated with our specific assays , are in favour of the usage of alternate dab-aegPNA in biomedical strategies, as we have in project to evaluate in continuing our research in the field of chiral nucleopeptide ODN analogs.

Acknowledgements

The authors would like to thank Dr Annalisa Cesarani and Dr Mariangela Castiglione for thoughtful discussions, and Mr Cristian D’Alessandro and Leopoldo Zona for their invaluable technical assistance.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Synthetic scheme of solid phase oligomerization and LC-ESIMS relative to the alternate dab-aegPNA t₁₂. See DOI: 10.1039/b910278g

‡ Abbreviations: CD (circular dichroism), DMEM (Dulbecco’s Modified Eagle’s Medium), DMF (N,N-dimethylformamide), FBS (fetal bovin serum), Fmoc (9-fluorenylmethoxycarbonyl), GAPDH (human glyceraldehyde 3 phosphate dehydrogenase), HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), HeLa (Epithelial cells from human adenocarcinoma), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), NMP (4-methylpyrrolidone), PTFE (polytetrafluoroethylene), RP (reverse phase), TAE (Tris-Borate-EDTA), TFA (trifluoroacetic acid), TMP (2,4,6-trimethylpyridine).

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