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

Abstract

The incorporation of C5-amino-modified 2′-deoxyuridine analogues into DNA have found application in nucleic acid labelling, the stabilization of nucleic acid structures, functionalization of nucleic acid aptamers and catalysts, and the investigation of sequence-specific DNA bending. In this study, we describe the physicochemical properties of four different C5-amino-modified 2′-deoxyuridines in which the amino group is tethered to the base via a 3-carbon alkyl, Z- or E-alkenyl or alkynyl linker. Conformational parameters of the nucleosides and their pKa values were deduced using 1H NMR. All of them display the expected anti-conformation of the nucleoside with 2′-endo sugar puckers for the deoxyribose ring. A preference for the cisoid conformation for the Z-alkenyl analogue is found, while the E-alkenyl analogue exists exclusively as its transoid conformation. The pKa values range from 10.0 for the analogue with an aliphatic propyl linker to 8.5 for the propargylamino analogue. The analogues have been used for the synthesis of triple-helix forming oligonucleotides (TFOs) in which they replace thymidine in the natural sequence. Oligonucleotides containing the propargylamino analogue display the highest stability especially at low pH, while those containing analogues with propyl and especially Z-alkenyl linkers are destabilized to a great extent. TFOs containing the analogue with the E-alkenyl linker have stability similar to the unmodified structures. The chemical synthesis of TFOs containing the analogue, 5-(3-hydroxyprop-1-ynyl)-2′-deoxyuridine that possesses a neutral but polar side chain show a remarkable stability, which is higher than that of all TFOs containing the alkylamino or alkenylamino analogues and only slightly lower than that of TFOs containing the propargylamino analogue. Both the hydroxyl and propargylamino substitutions impart enhanced triple-helix stability relative to the analogous sequences containing C5-propynyl-2′-deoxyuridine. Furthermore, a similar dependence of stability on pH is found between TFOs containing the hydroxypropynyl modifications and those containing the propargylamino side chains. This suggests that the major factor responsible for stabilizing such triple helices is due to the presence of the alkyne with an attached electronegative group.

2′-O-Dimethylaminoethoxyuridine and 5-Dimethylaminopropargyl Deoxyuridine for at Base Pair Recognition in Triple Helices

“…[8] Our results confirm this and show that TFOs with a positive charge attached to a propynyl group in the 5-position of thymine analogues are more stable than those with uncharged propynyl dU, even in the case of the relatively bulky dimethylamino group. It appears that hydrogen bonding is not the sole determinant of stability as the dimethylaminopropargyl substituent enhances triplex stability (although to a lesser extent than aminopropargyl).…”

Efficient Sequence-Directed Psoralen Targeting Using Pseudocomplementary Peptide Nucleic Acids

“…Many examples of such agents based on DNA minor groove binders (1), triple helix forming oligonucleotides (2), or zinc-finger DNA binding proteins (3) in combination with DNA alkylating reagents (4,5), photoreactive psoralens (6)(7)(8)(9), or nucleases (10, 11) have been described.…”

Hydroxyl-Functionalized DNA: An Efficient Orthogonal Protecting Strategy and Duplex Stability

“…Compared to the amino group (of 3-aminopropynyl), [23,24] the hydroxyl group is a neutral group, which makes its contribution less than aminopropynyl to the duplex and triplex stability. [13] When the hydroxyl group was linked to the nucleobase by alkyl chains, as shown in Table 3, the incorporation of the base pairs 1-dA, 2-dA, or 3-dA in place of dT-dA, from one to three incorporations, led to the decrease of the T m values, and more incorporations resulted in lower T m . The effect of these hydroxyalkyl groups was contrary to that of the rigid planar 3-hydroxypropynyl, probably because these alkyl linkers were flexible and not contributable to base stacking, and their hydrophobic property was not compatible with the hydrophilic environment of the major groove.…”