A Click Chemistry Approach to Targeted DNA Crosslinking with cis‐Platinum(II)‐Modified Triplex‐Forming Oligonucleotides

Abstract

Limitations of clinical platinum(II) therapeutics include systemic toxicity and inherent resistance.M odern approaches,t herefore,s eek new ways to deliver active platinum(II) to discrete nucleic acid targets.I nt he field of antigene therapy, triplex-forming oligonucleotides (TFOs) have attracted interest for their ability to specifically recognise extended duplex DNAtargets.Here,wereport aclickchemistry based approach that combines alkyne-modified TFOs with azide-bearing cis-platinum(II) complexes-based on cisplatin, oxaliplatin, and carboplatin motifs-to generate al ibrary of Pt II -TFO hybrids.T hese constructs can be assembled modularly and enable directed platinum(II) crosslinking to purine nucleobases on the target sequence under the guidance of the TFO.B yc ovalently incorporating modifications of thiazole orange-a knownD NA-intercalating fluorophore-into Pt II -TFOs constructs,e nhanced target binding and discrimination between target and off-target sequences was achieved.

Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking

“…20 Furthermore, applications of thiazole orange include its use as a surrogate base in PNA hybrids. 21 Whilst previous iterations of click Pt(II)-TFO hybrids displayed target destabilisation, 9 in the current work, we demonstrate that introduction of the AP-C3-dT monomer and TO B6 significantly enhances the stability of Pt(II)-TFO hybrids by mitigating the destabilising effects of platinum(II) crosslinking while simultaneously serving as a reporter for triplex formation. This study investigates the incorporation of a platinum drug and a fluorogenic intercalating probe on a single nucleobase.…”

“…The minor destabilisation incurred by TFO1-Carbo is consistent with our previous findings that cis-Pt(II)-TFO hybrids induce crosslinkdependent destabilisation of the underlying duplex target which also affects triplex formation. 9 Both TFO1 and TFO1- Carbo (Fig. 3A) failed to form stable triplexes at pH 6.5, 6.8, and 7.0.…”

“…The platinum agent cis-[Pt(2-azidopropane-1,3-diamine) (CBDCA)] (Pt-N 3 -Carbo) was synthesised as previously described. 9 Briefly, starting from K 2 PtCl 4 , Pt-N 3 -Carbo was isolated using a three-step procedure with 2-azidopropane-1,3-diamine as the azide-modified ligand. Thiazole orange (TO B6 ) was generated as reported by Klimkowski et al 15 The benzothiazole derivative was selected instead of the quinoline derivative (TO Q6 ) 18,22 as it is known to have a greater stabilising effect within DNA triplexes.…”

“…2 In recent years, antigene technology 3 -specifically triplex-forming oligonucleotides (TFOs) [4][5][6] -has emerged as an ideal vector for directing traditional platinum agents. [7][8][9][10] These modern strategies target extended genomic tracts while overcoming shortcomings in the targeting limitations of small molecule platinum drugs. 11 Precise targeting is the key impact of this technology with platinum nucleic acid hybrids achieving effective gene-knockdown in vitro.…”

“…These hybrids displayed targeting affinity combined with efficient triplex-mediated crosslinking. 9,12 Herein, we report the development of enhanced combination probes that facilitate both the click chemistry modification of TFOs with an azide-functionalised carboplatin analogue (Pt-N 3 -Carbo), and the coupling of a thiazole orange (TO B6 ) [13][14][15] fluorogenic intercalator. Click chemistry has recently been employed as an effective method for the direction and detection of azide-appended Pt(II) metallodrugs.…”