Stable DNA Triple Helix Formation Using Oligonucleotides Containing 2‘-Aminoethoxy,5-propargylamino-U

Abstract

We have prepared oligonucleotides containing the novel base analogue 2'-aminoethoxy,5-propargylamino-U in place of thymidine and examined their ability to form intermolecular and intramolecular triple helices by DNase I footprinting and thermal melting studies. The results were compared with those for oligonucleotides containing 5-propargylamino-dU and 2'-aminoethoxy-T. We find that the bis-substituted derivative produces a large increase in triplex stability, much greater than that produced by either of the monosubstituted analogues, which are roughly equipotent with each other. Intermolecular triplexes with 9-mer oligonucleotides containing three or four base modifications generate footprints at submicromolar concentrations even at pH 7.5, in contrast to the unmodified oligonucleotide, which failed to produce a footprint at pH 5.0, even at 30 microM. UV- and fluorescence melting studies with intramolecular triplexes confirmed that the bis-modified base produces a much greater increase in T(m) than either modification alone.

Four base recognition by triplex-forming oligonucleotides at physiological pH

“…Phosphoramidite monomers and other reagents were purchased from Applied Biosystems or Link Technologies. Phosphoramidites for BAU (41,42), Me P (16), S (30) and A PP (46) were prepared as described previously. The deprotected oligonucleotides were purified by reversed-phase high-performance liquid chromatography on a Brownlee Aquapore column (C8) using a gradient of acetonitrile in 0.1 M ammonium acetate.…”

“…The thermal melting temperature of the triplexes was determined using the fluorescence melting technique that we have developed previously (47) and have used for assessing the stability of triplexes that contain modified nucleotides (41,42,45,46). The third strand oligonucleotide is labelled at the 5′ end with a quencher (methyl red), while the 5′ end of the purine-rich strand of the duplex is labelled with a fluorescent group (fluorescein).…”

“…We have, therefore, examined the ability of a TFO containing four different modified nucleosides (BAU, Me P, A PP and S; see Figure 1A) to selectively target a mixed sequence at physiological pHs. BAU forms a very stable triplet with AT (41,42); Me P has a p K a that is higher than cytosine and targets GC base pairs at higher pHs (14–16); S has been proposed for recognizing TA inversions (30,45), while A PP recognizes CG (46). …”

Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

“…23 Attempts have been made to modify bases in the TFOs to tolerate pyrimidine interruptions [24][25][26][27][28] and to increase binding kinetics. 29 However, a general triple helix-based sequence recognition is not available.…”

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

“…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].…”