pH‐Independent Recognition of the dG ⋅ dC Base Pair in Triplex DNA: 9‐Deazaguanine N⁷‐(2′‐Deoxyribonucleoside) and Halogenated Derivatives Replacing Protonated dC

Abstract

Triplex-forming oligonucleotides (TFOs) containing 9-deazaguanine N 7 -(2'-deoxyribonucleoside) 1a and halogenated derivatives 1b,c were synthesized employing solid-phase oligonucleotide synthesis. For that purpose, the phosphoramidite building blocks 5a -c and 8a -c were synthesized. Multiple incorporations of 1a -c in place of dC were performed within TFOs, which involved the sequence of five consecutive 1a -c · dG · dC triplets as well as of three alternating 1a -c · dG · dC and dT · dA · dT triplets. These TFOs were designed to bind in a parallel orientation to the target duplex. Triplex forming properties of these oligonucleotides containing 1a -c in the presence of Na + and Mg 2+ were studied by UV/melting-curve analysis and confirmed by circular-dichroism (CD) spectroscopy. The oligonucleotides containing 1a in the place of dC formed stable triplexes at physiological pH in the case of sequence of five consecutive 1a · dG · dC triplets as well as three alternating 1a -c · dG · dC and dT · dA · dT triplets. The replacement of 1a by 9-halogenated derivatives 1b,c further enhanced the stability of DNA triplexes. Nucleosides 1a -c also stabilized duplex DNA. Essentially two families of DNA triple helices have been characterized i) those containing the pyrimidine · purine · pyrimidine motif, and ii) others forming a purine · purine · pyrimidine scheme. These two families differ in their third-strand composition and orientation. In the more commonly described pyrimidine · purine · pyrimidine motif, the third pyrimidine strand has been shown to bind within the major groove of duplex DNA in a parallel orientation with respect to the purine strand to form the triple helix. In this motif, the third-strand recognition occurs by the formation of dT · dA · dT and dCH + · dG · dC base triades through Hoogsteen pairing [2] [3]. The necessary protonation of cytosine limits the use of this method. Much effort has been devoted to increase the stability of triplexes under neutral conditions. The replacement of cytosine by 5-methylcytosine increases the stability of triplexes but does not alleviate the pH dependence [11 -14]. Oligonucleotides containing N

pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

“…According to this, the 9-propynyl substituent increases the stability of the amino protecting group similar to the 9-halogen substituents. 19 As the conformational freedom of the glycosylic bond could be restricted in compound 3b, 1 at the N-glycosylic bond, which is similar to the 9-halogenated derivatives. 17 According to the calibration graph for the estimation of the syn and anti conformer populations of b-D-nucleosides, 31 compound 3b gave 55% of an anti-rotamer population.…”

“…Up to now, we have studied triplex formation with systems containing three separate strands. 19 Now we are investigating triplex formation on the hairpin triplex 21 being related to a 31-mer hairpin. 10 Compared to a triplex formed by three individual oligonucleotides, such an intramolecular triplex is more stable.…”

“…At first we incorporated the 9-deazaguanine N 7 -(2deoxyribonucleoside) 3a 19 which resembles the recognition site of a protonated 2 -deoxycytidine in position X and in positions X and Y of the oligonucleotide 21. As the propynyl group is a standard residue to stabilize DNA-duplexes 26 we reasoned that these favorable properties can also be seen on triplexes.…”

pH-Dependent Assembly of DNA–Gold Nanoparticles Based on the i-Motif: A Switchable Device with the Potential of a Nanomachine

“…The oligonucleotides were synthesized in an automated DNA synthesizer (ABI 392, Applied Biosystems, Weiterstadt, Germany) at a 1-mmol scale according to the standard protocol for 3'-(2-cyanoethyl phosphoramidites) employing the 5'-O-[(tritylthio)hexyl] modifier for the oligonucleotides 3 and 4 [27]. Deprotection and purification of the unmodified oligonucleotides 1 and 2 was performed as described earlier [20]. The modified oligonucleo- tides 3 and 4 were deprotected with 25% aq.…”

“…The oligonucleotide syntheses were performed in an automated DNA synthesizer by means of solid-phase synthesis. The purification followed the standard protocol [20], and the oligomers were characterized by MALDI-TOF mass spectra. The calculated masses were in good agreement with the measured values (Table).…”