Use of a pyrimidine nucleoside that functions as a bidentate hydrogen bond donor for the recognition of isolated or contiguous G-C base pairs by oligonucleotide-directed triplex formation

Abstract

Synthesis of the nucleoside building block of the 6-keto derivative of 2'-deoxy-5-methylcytidine (m5oxC) as an analog of an N3-protonated cytosine derivative is described. A series of 15mer oligonucleotides containing either four or six m5oxC residues has been prepared by chemical synthesis. Complexation of the 15 residue oligonucleotides with target 25mer duplexes results in DNA triplexes containing T-A-T and m5oxC-G-C base triplets. When the m5oxC-G-C base triplets are present in sequence positions that alternate with TAT base triplets, DNA triplexes are formed with Tm values that are pH independent in the range 6.4-8.5. A 25mer DNA duplex containing a series of five contiguous G-C base pairs cannot be effectively targeted with either m5C or M5oxC in the third strand. In the former case charge-charge repulsion effects likely lead to destabilization of the complex, while in the latter case ineffective base stacking may be to blame. However, if the m5C and M5oxC residues are present in the third strand in alternate sequence positions, then DNA triplexes can be formed with contiguous G-C targets even at pH 8.0.

Targeting DNA with Triplexes

“…The less efficient base stacking observed with this analogue is consistent with the observation that this base does not form stable triplexes at blocks of contiguous guanines, even though it does not suffer from the charge repulsion seen with C + and 5Me C + [33]. Surprisingly stable recognition of G-tracts is achieved by alternating this base analogue with 5Me C [33]. More recently it has been shown that 6-oxocytosine forms a more stable triplet when deoxyribose is replaced with a flexible acyclic linker [34].…”

“…This could reflect the importance of the positive charge noted above, affecting the energetics of base stacking, or might be due to changes in the hydrogen bonding characteristics since the natural bases form one hydrogen bond with a charged partner in contrast to the uncharged partner with the synthetic base. The less efficient base stacking observed with this analogue is consistent with the observation that this base does not form stable triplexes at blocks of contiguous guanines, even though it does not suffer from the charge repulsion seen with C + and 5Me C + [33]. Surprisingly stable recognition of G-tracts is achieved by alternating this base analogue with 5Me C [33].…”

“…(2c) is a further pyrimidine analogue which presents the correct arrangement of hydrogen bonds for pH independent recognition of guanine [31][32][33][34]. Although this base forms a pH independent triplet with GC, the complexes generated are less stable than those observed with C or 5Me C at low pH.…”

“…Pyrimidine analogues for recognition of GC base pairs in a parallel triplex. a) the 5Me C + q GC triplet, b) pseudoisocytosine [27,28], c) 6-oxocytosine [31][32][33][34], d) pyrazine analogue [37], e) 2-aminopyridine [38][39][40][41]. In b) -e) the third strand base analogue is shown alone in the same orientation as in a), indicating the substituents that form hydrogen bonds to G. Purine analogues for recognition of GC base pairs in a parallel triplex.…”

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

“…We then examined the formation of triplexes with oligonucleotides containing 15 base pairs with multiple incorporations of the modified nucleosides, both involving either a consecutive run of noncanonical residues or those following an alternating scheme. The target duplexes 17 · 18 and 19 · 20 shown in Table 3 follow the same sequence which has been already used by McLaughlin and co-workers [37]. The third strands containing the dCH + · dG · dC motif is expected to bind in parallel orientation to the target duplex by Hoogsteen base pairing.…”