Tandem trimer pyrrole-imidazole polyamide probes targeting 18 base pairs in human telomere sequences

Abstract

The binding of molecules to specific DNA sequences is important for imaging genome DNA and for studying gene expression. Increasing the number of base pairs targeted by these molecules would provide greater specificity. N-Methylpyrrole-N-methylimidazole (Py-Im) polyamides are one type of such molecules and can bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. Our recent work has demonstrated that tandem hairpin Py-Im polyamides conjugated with a fluorescent dye can be synthesized easily and can serve as new probes for studying human telomeres under mild conditions. Herein, to improve their selectivities to telomeres by targeting longer sequences, we designed and synthesized a fluorescent tandem trimer Py-Im polyamide probe, comprising three hairpins and two connecting regions (hinges). The new motif bound to 18 bp dsDNA in human telomeric repeats (TTAGGG) _n , the longest sequence for specific binding reported for Py-Im polyamides. We compared the binding affinities and the abilities to discriminate mismatch, the UV-visible absorption and fluorescence spectra, and telomere staining in human cells between the tandem trimer and a previously developed tandem hairpin. We found that the tandem trimer Py-Im polyamide probe has higher ability to recognize telomeric repeats and stains telomeres in chemically fixed cells with lower background signal.

Fig. 1. Chemical structures of Py–Im polyamides TH59, TT59, TAMRA TH59 and TAMRA TT59 targeting the human telomere sequence and their ball-and-stick representations.

Scheme 1. Solid-phase synthesis of Py–Im polyamides TH59, TT59, TAMRA TH59 and TAMRA TT59 and structure of building block 1. Reagents and conditions: (i) 20% piperidine, NMP; (ii) Fmoc–PyIm–CO₂H, HCTU, DIEA, NMP; (iii) 20% piperidine, NMP; (iv) Fmoc–Py–CO₂H, HCTU, DIEA, NMP; (v) 20% piperidine, NMP; (vi) Fmoc–Py–CO₂H, HCTU, DIEA, NMP; (vii) 20% piperidine, NMP; (viii) 1, HCTU, DIEA, NMP; (ix) 20% piperidine, NMP; (x) Fmoc–mini-PEG–OH, HCTU, DIEA, NMP; (xi) 20% piperidine, NMP; (xii) Fmoc–PyIm–CO₂H, HCTU, DIEA, NMP; (xiii) 20% piperidine, NMP; (xiv) Fmoc–Py–CO₂H, HCTU, DIEA, NMP; (xv) 20% piperidine, NMP; (xvi) Fmoc–Py–CO₂H, HCTU, DIEA, NMP; (xvii) 20% piperidine, NMP; (xviii) 1, HCTU, DIEA, NMP; (xix) 20% piperidine, NMP; (xx) 3,3′-diamino-N-methyldipropylamine, 55 °C; (xxi) 5-carboxytetramethylrhodamine, succinimidyl ester, DIEA, DMF.

Fig. 2. Telomere staining of HeLa 1.3 cell spreads with fluorescent polyamides. (A) The HeLa 1.3 cell spread was stained with the fluorescent polyamide (first row) and DAPI (second row). The merged images are shown in the third row. The first and second columns show the results for TAMRA TH59 and TAMRA TT59, respectively. Enlarged images of the boxed regions in panel (A) are shown in panel (B).

Fig. 3. Telomere staining of HeLa 1.3 cells with fluorescent polyamides. HeLa 1.3 cells were stained with DAPI (first column) and fluorescent polyamides (second column). The merged images are shown in the third column. Enlarged images of the boxed regions in the first row are shown in the second row. Cell images using TAMRA TH59 and TAMRA TT59 are shown in panels (A) and (B), respectively.

Fig. 4. (A) Surface plots based on the boxed images shown in Fig. 3. (B) Relative signal-to-noise ratios of the images.