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

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.


Instrumentation
Reversed-phase HPLC analysis and purification were performed on Agilent Technologies 1200 analytical and preparative chromatographs piloted by the Agilent ChemStation program. Mass-spectrometry analysis by electrospray Q-TOF (ESI Q-TOF MS) was performed on a Q-Star instrument (Applied Biosystems, Courtaboeuf, France) in the positive (for polyamides) or negative (for oligonucleotides) mode; analyses were performed in methanol or water, respectively. Polyacrylamide gels with fluorescently labeled oligonucleotides and conjugates were scanned on a s3 Typhoon 9410 (Amersham) fluorescence scanner with laser excitation at 532 nm and emission filter at 526 nm. Non-labeled oligonucleotides and their conjugates were detected after polyacrylamide gel-electrophoresis by "UV-shadowing" method on fluorescent screen in SynGene G-box gel imaging system. UV-vis spectra and thermal denaturation curves were obtained on a UV-visible spectrophotometer "Uvicon XL" (Secomam, Nova Analytic Company) with 12 quartz cells installed in a mobile thermostated support piloted by ThermAlys software (DuDoTec GmbH).
Fluorescent TINA oligonucleotides were visualized in GBox by fluorescence on Transilluminator with excitation wavelength 302 nm. NMR spectra were recorded on Bruker AVANCE 400 NMR spectrometer equipped with a 1 H broadband reverse gradient probe head.

Coupling of the azide-bearing fluorophore group to the alkynemodified polyamide
The thiazol-3-ium (TO, thiazole orange) has been described in [9].

Synthesis of TFOs 15-19 bearing terminal alkynes
Modified oligonucleotides, bearing one or two alkyne linkers at the 5'-end, were prepared by Mukaiyama reaction [10] Table S2.  The yields were 30-90 % (see Tables S3 and S4).  The most deceiving surprise was a rapid degradation of the HPLC-purified conjugates even after one-day storage in the freezer ( Figure S1). This resin allowed us to remove copper ions completely and to save the newly synthesized TINA-TFO-polyamide conjugates from decomposition.

Synthesis of polyamide-TFO conjugates (23-29) by CuAAC reaction
As an alternative method of purification we also tried a preparative denaturing gel electrophoresis in 1 mm thick 20% polyacrylamide geltris-borate-EDTA -7 M urea. Here, the good resolution of conjugates, initial products and degradation products has been observed. After excision of the product band and water extraction, a homogeneous pure conjugate was obtained. However, the yield of this procedure was quite low (about 20%).

Gel-shift experiments
Gel-shift experiments were performed in 15% non-denaturing polyacrylamide gels in 50 mM HEPES (pH 7.2), 50 mM NaCl and 5 mM MgCl 2 . Each sample (10 µL) in the same buffer contained 1.2 pmol of fluorescein-labeled target HIV duplex with covalently linked complementary strands via four thymidine nucleotides (see publication [12]), glycerol (up to 8%) and eventually 0.06% bromophenol blue and xylene cyanol as migration markers. The mixture was first heated at 90 °C for 3 min and slowly cooled, then the third strand (conjugates 23-29) was added up to its specific final concentration in each tube (0.45-3 µM). The mixtures were incubated at +4 °C overnight and then loaded onto 15% non-denaturing polyacrylamide gel. The electrophoresis was performed at a power 5 W for several hours. The gels were scanned on Typhoon using a laser at 473 nm and a 510LP filter to detect the fluorescein label. The image was analyzed using the ImageQuant or ImageJ s10 software. Apparent dissociation constants were calculated according to the protocol described in publication [12].