Cross-linking to an interrupted polypurine sequence with a platinum-modified triplex-forming oligonucleotide

Abstract

Triplex-forming oligonucleotides (TFOs) can bind specifically to polypurine sequences in doublestranded DNA. A single interruption of this polypurine tract can greatly destabilize triplex formation. The stability of triplexes can be significantly enhanced by covalently linking the TFO to its DNA target with reactive functional groups conjugated to the TFO. Covalently cross-linked TFOs are effective inhibitors of transcription of the target DNA sequence. We have designed a TFO with a platinum-modified base that can interact with and cross-link to a cytosine interruption in the polypurine tract of a target DNA duplex. The TFO contains an N 4 -(aminoalkyl)cytosine derivatized with cis-or trans-diamminediaquaplatinum(II). When bound to its target, the tethered platinum of the TFO can reach across the major groove and form an adduct with the guanine N7 of the interrupting C·G base-pair. The optimal tether length is five methylene groups, and cross-linking is most efficient when the tether is modified with trans-diamminediaquaplatinum. Cross-linking requires that the TFO is bound to its designated DNA target. Addition of cyanide to the cross-linked TFO product reversed the cross-link, behavior that is consistent with the presence of a Pt-guanine adduct. The kinetics of the cross-linking reaction were studied and the half-life of the cross-linking reaction was approximately three hours. Our results demonstrate that platinum-conjugated TFOs can be designed to cross-link with DNA targets that contain a single pyrimidine interruption. Modifications of this type may prove useful for expanding the DNA sequences that can be targeted by TFOs and increasing the stability of the resulting triplexes.

TTS Mapping: integrative WEB tool for analysis of triplex formation target DNA Sequences, G-quadruplets and non-protein coding regulatory DNA elements in the human genome

“…Only a single pyrimidine insertion can greatly decrease triplex stability [ 15 , 23 ]. Although modified TFOs can overcome the limitation, the insertions still affect triplex stability in these cases [ 24 ]. Nevertheless, a few pyrimidine insertions still have to be considered.…”

Transplatin-Conjugated Triplex-Forming Oligonucleotides Form Adducts with Both Strands of DNA

“…The sequences of these TFOs and the site targeted for triplex formation are shown in Table . The target duplex sequence (4RY) serves as a model for triplex formation with five cytosine residues within a 17 bp long polypurine tract and is similar to a previously studied purine target sequence found in the coding region of the polymerase gene of HIV-1 , . Each of the TFOs consisted of either 2′-deoxyribonucleotides or 2′- O -methylribonucleotides and contained 5-methylcytosine ( C ) and 5-methyluracil (T) bases.…”

“…The primary target of cisplatin is guanine, and cisplatin can form both intrastrand and interstrand cross-links in DNA. Platinum compounds have been employed in several ways to cross-link TFOs to duplex DNA including tethering cisplatin to the 5′-end of a TFO , , targeting an interruption in the polypurine tract with a transplatin-modified cytosine residue , , and using transplatin monoadducts of guanine and cytosine to react within the Hoogsteen triad , .…”

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

“…In comparison to existing methods used to prepare platinated TFOs, click chemistry affords a modular approach whereby the incorporation of cis ‐platinum(II) at practically any location within the probe strand is possible. This approach also overcomes limitations that require complexation between a platinum(II) reagent, such as a trans ‐platinum(II) complex,[ 10a , 10b ] and the TFO substrate—an approach that hitherto precluded the development of cis ‐platinum(II)‐type hybrids [14a] and the ability to generate 1,2‐d(GpG) cisplatin lesions central to their clinical success. The use of PAGE analysis indicates that, for several TFO constructs, numerous platinated triplex adducts can form, giving rise to distinctive banding patterns that are reversed upon removal of the crosslink with sodium cyanide.…”