Solution Structure of an Intramolecular DNA Triplex Containing 5-(1-Propynyl)-2‘-deoxyuridine Residues in the Third Strand^,

Abstract

Incorporation of the modified base 5-(1-propynl)-2'-deoxyuridine (propynylU) in the third strand of a triplex leads to enhanced triplex stabilization. To investigate effects of the propyne nucleotide on triplex structure and the factors underlying the increased stability, we have determined the solution structure of the intramolecular DNA pyrmidine-purine-pyrimdine d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-PCPCPCPP) (PDD-EG), which contains 5-(1-propynl)-2'-deoxyuridine (P) in the third strand and hexakis(ethylene glycol) linkers [(EG)6]. The structure was calculated using X-PLOR with distance and dihedral angle restraints obtained from two-dimensional NMR experiments and refined with the direct relaxation matrix method. The structures show that the extended aromatic electron cloud of the propynylU nucleotide stacks well over the 5'-neighboring nucleotides, resulting in increased stabilization. The propynylU nucleotides also affect the overall structure of the triple helix. A comparison of the structure to that of the nonmodified intramolecular DNA triplex of the same sequence, d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-TCTCTCTT) (DDD-EG), shows that PDD-EG has a more A-DNA like X displacement and inclination than DDD-EG yet still maintains predominantly S-type sugar puckers as found in DDD-EG and other DNA triplexes.

Targeting DNA with Triplexes

“…A similar effect is seen with 5-propynyl-U, Fig. (5a), which also increases triplet stability [73][74][75], though 5-propynyl-C produces a less stable triplet since this substitution further reduces the pK of N3 to 3.3. The increased stability of 5-propynyl substituted bases is presumably due to increased base stacking interactions.…”

“…However, simple addition of aromatic rings to the pyrimidine nucleus does not seem to be of benefit [76][77][78][79], possibly because the improved stacking of the single stranded oligonucleotide hinders triplex formation. Other stacking interactions, such as those involving appended propynyl or propargylamino groups [72][73][74][75], may be a better option. Lastly it is possible that different sugars should be used for third strand C or T, since it appears that rigid linkers improve the stability of T q AT, while flexible linkers improve the stability of C +q GC [35,36].…”

Amino-functionalized DNA: the properties of C5-amino-alkyl substituted 2'-deoxyuridines and their application in DNA triplex formation

“…The ability of C5-propynyl-2′-deoxyuridine (i.e. with no appended cations to the nucleobase) to stabilize triple helices through enhanced base stacking is well-documented (36,37). In DNA duplexes and triplexes, a C5-propynyl-modified cytosine or uracil can enhance stability (7).…”

Selectivity and affinity of DNA triplex forming oligonucleotides containing the nucleoside analogues 2′-O-methyl-5-(3-amino-1-propynyl)uridine and 2′-O-methyl-5-propynyluridine

“…In our earlier work we found that a patch of 3-4 contiguous 2′-AE residues made an important contribution towards the bioactivity of TFOs containing 2′-O-Me ribose sugars 23,30. Accordingly we synthesized TFO- 15 with 2′-O-Me ribose, four contiguous 2′-AE residues at the 3′ end, and the alternating distribution pattern of 2 found in TFO- 12 .…”

“…However, when we determined the k on value for TFO- 15 we found a pronounced decline relative to the TFO- 14 control. This was unexpected since both the 2′-AE cluster,30 and the alternating pattern of 2 (Table 2) enhance association rate relative to TFOs containing only the 2′-O-Me modification. Given the importance of the association rate as a correlate of TFO bioactivity, we examined the properties of two additional TFOs containing 2′-AE and 2′-O-Me ribose.…”