Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

Abstract

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.

Graphical Abstract

Acridine-4-carboxamides (both freely in solution and attached covalently to the probe) can increase thermal stability of short oligodeoxynucleotide probes leading to good discrimination of single-based mismatches.

Figure 1.

Published acridine derivatives used for modification of DNA.

Scheme 1.

Synthesis of acridine modifiers. Reaction conditions: i) Cu⁰, K₂CO₃, isopentyl alcohol, aniline (for 13) or anthranilic acid (for 14), reflux overnight; ii) 1. 13, POCl₃, reflux, 1 h; 2. phenol, 110°C, 15 min; 3. 6-azidohexyl-1-amine, 55°C, overnight; iii) 1. 14, POCl₃, reflux, 1 h; MeOH, 50°C, overnight; iv) 1. SOCl₂, 80°C, 1 h; 2. phenol, 110°C, 15 min; 3. 6-azidohexyl-1-amine, 55°C, overnight; v) THF, excess of saturated solution of NaOH in MeOH:H₂O 5:1, 50°C, 1 h; vi) 1. SOCl₂, rt, 1 h; 2. Amine (1 equiv.), TEA (4 equiv.), DCM, 0°C, 30 min. Compounds 2–12, except compound 9, were converted to corresponding hydrochlorides.

Scheme 2.

(A) Schematic illustration of the modified probes. (B) Principle of ‘click’ modification of the probes on solid phase support. Dark-green dot–FAM, red dot–DBCO, grey dot–solid phase support, yellow stick–acridine modifier. S refers to ‘short’ and L to ‘long’ probe having 13 and 18 bases, respectively; numbers 1, 7 or 13 indicate the position of the acridine in the ODN probe.

Chart 1.

Sequences of the probes, the controls, and the targets used in the study. Probes contain DBCO-modified T-base used for ‘click’ modification. * Probes without FAM labelling. S– short (13 bases), L– long (18 bases) ODNs. Numbers 1, 7 and 13 indicate the position of the acridine in the ODN probe. Phos = phosphate.

Figure 2.

(A) Chromatogram of S_7_2 (λ = 260 nm). (B) Absorption spectra of two peaks at chromatogram (A). (C) Mass spectrum of S_7_2 probe.

Figure 3.

(A) Melting curves of the duplex C_L–T_L alone (black) or with selected acridine derivatives, 2 (blue), 4 (red) and 11 (dashed green) at concentration 6.25 μM. (B) Screening test of acridine derivatives in the HFE melting system in solution expressed as dependence of the change of ΔT_m on the concentration of acridine. Vertical dotted line is drawn at c = 6.25 μM used for comparison purposes. The test was performed in triplicate. (C) Scheme of HFE melting system.

Chart 2.

ODNs duplexes used for study of interaction of acridine 2 with ODNs. Sense strands (used as ssDNAs in the study) are marked by a red panel.

Figure 4.

Changes in absorption (A–C) and emission (E–G) spectra (λ_ex = 380 nm) of acridine 2 (20 μM) during the titration by AT-rich (A, E), GC-rich (B, F), and HFE (C, G) duplexes in reaction buffer. (D, H) Changes in absorption at 417 nm (D) and emission at 530 nm (H) of acridine 2 during the titration by duplexes (AT-rich–green, GC-rich–red, HFE–blue), and ssGC-rich (purple). (I, J) Comparison of absorption (I) and emission (J) spectra of 2 (20 μM) complexed to AT-rich (green), GC-rich (red) and HFE (blue) duplexes (20 μM). (K) Schematic principle of the experiment.

Figure 5.

Job's plot of the interaction of 2 with (A) dsHFE, (B) ssHFE, (C) dsAT, (D) dsGC (monitored as absorption of acridine at 417 nm).

Figure 6.

(A) Comparison of melting curves and corresponding melting peaks (B) of the L_7_4 (blue), L_13_4 (red) in duplex with T_L and control unmodified duplex C_L–T_L without acridine moiety (black, dashed). (C) Comparison of melting curves and corresponding melting peaks (D) of L_13_4 matched to the target T_L (blue), L_13_4 hybridised to the A mismatched target T_L(A) (red), L_13_4 hybridised to the C mismatched target T_L(C) (green), and control unmodified perfectly matched duplex C_L–T_L without acridine moiety (black, dashed). All melting curves were measured at the channel for FAM (λ_em = 515–530 nm). All measurements were performed in triplicate.

Figure 7.

Determination of the potential mutations in the HFE gene in models of real samples. (A) Template containing HFE gene sequence. (B) Principle of the determination. (C) Melting curves and melt peaks (D) of the wild type of HFE (matched, blue) and its S65C A > T (red), and H63D C > G (green), determined with probe L_7_2. The control experiment with probe L_7 (no acridine conjugation) and wild type HFE perfectly matched is shown by the black dashed line.

Figure 8.

(A, C) Changes of absorption (A) and emission (C) spectra of L_7_2* probe titrated by T_L; (B) Comparison of absorption spectra of 2 bound to ssHFE (blue) or dsHFE (red) freely in solution (full lines). Dashed lines are absorption spectra of L_7_2* with (red) or without (blue) complementary T_L; (D) normalised changes in absorbance (blue) and fluorescence (red), of L_7_2* during titration with T_L (data from boxes A and C). Concentration of L_7_2* was 10 μM. T = 23°C.

Figure 9.

(A) Temperature dependent emission (λ_ex = 400 nm) changes of L_1_2* probe matched duplex with T_L. (B) Comparison of melting curves of the S_1_2* (blue) and C_S (black) in duplex with T_S. (C) Comparison of melting curves and melting peaks (D) of the S_1_2* matched to the target T_S (blue), S_1_2* hybridised to the A mismatched target T_S(A) (red), and S_1_2* hybridised to the C mismatched target T_S(C) (green).