DNA triplex formation with 5-dimethylaminopropargyl deoxyuridine

Abstract

We have prepared triplex-forming oligonucleotides containing the nucleotide analogue 5-dimethylaminopropargyl deoxyuridine (DMAPdU) in place of thymidine and examined their ability to form intermolecular triple helices by thermal melting and DNase I footprinting studies. The results were compared with those for oligonucleotides containing 5-aminopropargyl-dU (APdU), 5-guanidinopropargyl-dU (GPdU) and 5-propynyl dU (PdU). We find that DMAPdU enhances triplex stability relative to T, though slightly less than the other analogues that bear positive charges (T << PdU < DMAPdU < APdU < GPdU). For oligonucleotides that contain multiple substitutions with DMAPdU dispersed residues are more effective than clustered combinations. DMAPdU will be especially useful as a nucleotide analogue as, unlike APdU and GPdU, the base does not require protection during oligonucleotide synthesis and it can therefore be used with other derivatives that require mild deprotection conditions.

The stability of triplex DNA is affected by the stability of the underlying duplex

“…When these triplexes were heated and cooled at faster rates there was hysteresis between the melting and annealing profiles, as previously observed [23,30], indicating that one or more steps in the association or dissociation pathways are slow and that the system is not at thermodynamic equilibrium. Representative melting and annealing curves at a fast rate of temperature change (12 °C.min -1 ) are shown in Fig.…”

“…Triplex melting curves are commonly used to assess the stabilising effects of nucleotide substitutions [16,19,23,[30][31][32][33][34][35]. Such modifications can produce complexes in which the triplex and duplex melts overlap, producing a single melting transition.…”

“…These are usually performed with short synthetic duplex targets, which are of similar length to the third strand. In some experiments the duplex has been made longer than the triplex in order to separate the duplex and triplex melting transitions [21][22][23], while others have employed intramolecular duplexes in order to increase the stability of the target [24][25][26]. In contrast in most footprinting experiments and for in vivo applications the duplexes in which the target sequences are located will be much longer than the third strand [19,27,28].…”

Insights into the Relative DNA Binding Affinity and Preferred Binding Mode of Homologous Compounds Using Isothermal Titration Calorimetry (ITC)

“…[21][22][23][24][25] Stabilization of DNA triplexes formed when oligonucleotides (normally referred to as triplex formation oligonucleotide or TFO) bind to DNA duplexes, have been explored in anti-gene therapeutics where expression of deleterious DNA sequences are suppressed by the binding and stabilization of complimentary TFO sequences. [15,21] Formation of transient G-quadruplexes in G-rich sequences have been found to be prominent in telomeres, G-rich ends on chromosomes that protects indispensable genes from being depleted, as well as preventing unwanted chromosomal fusions.…”

“…[21][22][23][24][25] Stabilization of DNA triplexes formed when oligonucleotides (normally referred to as triplex formation oligonucleotide or TFO) bind to DNA duplexes, have been explored in anti-gene therapeutics where expression of deleterious DNA sequences are suppressed by the binding and stabilization of complimentary TFO sequences. [15,21] Formation of transient G-quadruplexes in G-rich sequences have been found to be prominent in telomeres, G-rich ends on chromosomes that protects indispensable genes from being depleted, as well as preventing unwanted chromosomal fusions. [23][24][25] As a result, some compounds (e.g., certain NDI derivatives) can bind to and stabilize these telomeric G-quadruplexes can block access to these sequences by telomerase enzymes, which are responsible for extending and protecting telomeres and have been found to be over-expressed in 80% of cancers cells.…”