Application of Cu(I)-catalyzed azide-alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA

Abstract

Efficient protocols based on Cu(I)-catalyzed azide-alkyne cycloaddition were developed for the synthesis of conjugates of pyrrole-imidazole polyamide minor groove binders (MGB) with fluorophores and with triplex-forming oligonucleotides (TFOs). Diverse bifunctional linkers were synthesized and used for the insertion of terminal azides or alkynes into TFOs and MGBs. The formation of stable triple helices by TFO-MGB conjugates was evaluated by gel-shift experiments. The presence of MGB in these conjugates did not affect the binding parameters (affinity and triplex stability) of the parent TFOs.

Keywords: Cu(I)-catalyzed azide–alkyne cycloaddition; binding affinity; click chemistry; pyrrole–imidazole polyamides; sequence specificity: DNA; triplex-forming oligonucleotides.

Figure 1

A) Formation of nucleotide triplets in parallel and antiparallel (relatively to polypurine strand) complexes. The relative orientations of DNA backbones are shown by the symbols “” and “”. The duplex is in green, TFO is in black. B) Chemical structure of the TINA molecule inserted in the TFO structure of the DNA triplex as a bulge.

Figure 2

Synthesis of MGB-fluorophore (A) and MGB-TFO (B) conjugates using CuACC. Linker length and composition can be varied.

Figure 3

Bifunctional linkers for conjugation of oligonucleotides and polyamides using CuACC.

Figure 4

The target duplex contains a 29 base pair fragment from HIV proviral DNA [35] and a T4 hairpin is connecting two strands: a 16 base pair polypurine-polypyrimidine tract (TFO-binding site – is indicated by a red rectangle), an adjacent overlapping poly(dA:dT) tract (MGB-binding site – by a blue rectangle). Fluo is a fluorescein label.

Figure 5

A) Sequence derived from the murine pericentromere repeat fragment with only one target site for the polyamide F1-NH₂ [–3]. The target sequence is underlined. B) Chemical formula of the polyamide F1-NH₂.

Figure 6

Synthesis of azide- and alkyne-modified MGBs.

Figure 7

Structures of fluorescent probes synthesized by "click chemistry".

Figure 8

Titration of the probes F1-NH₂-MM14 (12 µM, A, C) and F1-NH₂-TO (10 µM, B, D) by the target DNA duplex (left) and non-cognate duplex (right) in 0.025 M tris-HCl, pH 7.5, at room temperature. The sequence of the target duplex is depicted in Figure 5A, the sequence of the non-cognate duplex is 5'-GGGTGAGGAGGAGGGAGATCGGTC-3'/3’-CCCACTCCTCCTCCCTCTAGCCAGT-5'. DNA concentration after each addition is indicated on the right panel of each picture.

Figure 9

Synthesis of modified oligonucleotides containing an alkyne group.

Figure 10

Gel electrophoresis of oligonucleotides modified by alkyne linkers: A – oligonucleotide HIVP (detection by UV-shadowing). Line 1 – control, starting oligonucleotide, lines 2 and 3 – reaction mixtures after its interaction with linker 1 (product 18) and linker 2 (product 19), respectively. B – oligonucleotide HIVLP modified by linker 1. Line 4 – control, starting oligonucleotide, lines 5–7 – electrophoresis of individual products obtained after purification of reaction mixture by HPLC. 5 – initial oligonucleotide, 6 – first peak (product 15), 7 – second peak (product 16).

Figure 11

TINA-TFOs bearing a 3'-alkyne group for antiparallel triplex formation with the target HIV proviral DNA (Figure 4).

Figure 12

Structures of polyamide-TFO conjugates.

Figure 13

Electrophoresis analysis of samples from reaction mixtures after click reactions between alkyne-TFO and azide-polyamide in denaturing 20% polyacrylamide gel, visualization by UV-shadowing. A) conjugates 23 and 25: lane 1 – control oligonucleotide 15, lane 2 – conjugate 23, lane 3 – control oligonucleotide 17, lane 4 – conjugate 25. B, conjugate 24: lane 5 is control oligonucleotide 16, lane 6 – conjugate 24. The arrows indicate positions: a and c – initial alkyne-modified oligonucleotides, b and e – 1:1 conjugates TFO:polyamide, f – 1:2 conjugate TFO-polyamide, d – visible migration marker xylene cyanol.

Figure 14

Electrophoresis analysis of reaction mixtures in 20% denaturing polyacrylamide gel after TINA-TFO-MGB conjugation (visualization by TINA fluorescence). A) Conjugates 26 (lanes 2 and 3), 27 (lane 5), 29 (lane 7). Lanes 1, 4 and 6 are control oligonucleotides 20, 21 and 22, respectively B) Conjugate 28 (lane 9); lane 8 – control oligonucleotide 21. Arrows indicate: a, control oligonucleotides, b, conjugates.

Figure 15

Electrophoretic analysis of reaction mixtures in standard 20% denaturing PAGE after DNA-templated synthesis. All reaction mixtures in the 0.1 M TEA-acetate buffer, pH 7.0 contained 10 µM polyamide 14 and components, indicated in the table. The concentrations of the DNA template (target duplex shown in Figure 3) and TINA-TFOs (shown in Figure 10) were 10 µM. Cu(I) was added as a mixture of components up to the final concentrations of CuSO₄ and THPTA 5 mM and sodium ascorbate 10 mM.

Figure 16

Non-denaturing gel electrophoresis analysis of conjugate 28 with fluorescein-labeled target HIV duplex (concentration 120 nM), polyamide concentrations: 0, 0.45, 0.75, 1.13, 1.5, 2.25, 3, 0 μM (lines 1 to 8, respectively) in 0.05 M HEPES, 50 mM NaCl, 5 mM MgCl₂, pH 7.2. Visualization of the gel was performed by monitoring fluorescence of the fluorescein-labeled duplex depicted in Figure 4.