Abstract
Propargyl groups are attractive functional groups for labeling purposes, as they allow CuAAC-mediated bioconjugation. Their size minimally exceeds that of a methyl group, the latter being frequent in natural nucleotide modifications. To understand under which circumstances propargyl-containing oligodeoxynucleotides preserve base pairing, we focused on the exocyclic amine of cytidine. Residues attached to the exocyclic N4 may orient away from or toward the Watson–Crick face, ensuing dramatic alteration of base pairing properties. ROESY-NMR experiments suggest a uniform orientation toward the Watson–Crick face of N4-propargyl residues in derivatives of both deoxycytidine and 5-methyl-deoxycytidine. In oligodeoxynucleotides, however, UV-melting indicated that N4-propargyl-deoxycytidine undergoes standard base pairing. This implies a rotation of the propargyl moiety toward the ‘CH’-edge as a result of base pairing on the Watson–Crick face. In oligonucleotides containing the corresponding 5-methyl-deoxycytidine derivative, dramatically reduced melting temperatures indicate impaired Watson–Crick base pairing. This was attributed to a steric clash of the propargyl moiety with the 5-methyl group, which prevents back rotation to the ‘CH’-edge, consequently preventing Watson–Crick geometry. Our results emphasize the tendency of an opposing nucleic acid strand to mechanically rotate single N4-substituents to make way for Watson–Crick base pairing, providing no steric hindrance is present on the ‘CH’-edge.
Highlights
The chemical modification of nucleic acid monomers has become common practice in a variety of research fields for gaining access to artificially enhanced DNA and RNA properties
To investigate the orientation of a propargyl group on the exocyclic N4 of cytidines on both the nucleoside level and in an ODN context, we devised a synthetic route to a common precursor, from which both the free nucleoside and the phosphoramidite required for ODN synthesis could each be obtained in a single reaction step
We recorded melting curves of duplexes of selected oligonucleotides of the ODNx.1 series, with DNA strands featuring abasic sites opposite the selected modified positions: ODN10.1 was hybridized to COMP10 and ODN11.1/11.2 were hybridized to COMP11 (Figure 4). These correspond to duplexes lacking the opposing guanosine, which leaves no Watson–Crick partner, but provides space to alleviate a steric clash caused by the propargyl group
Summary
The chemical modification of nucleic acid monomers has become common practice in a variety of research fields for gaining access to artificially enhanced DNA and RNA properties. [3+2] cycloaddition reactions between azides and alkynes (CuAAC and SPAAC), together with other forms of click reactions, are amongst the favorites of the bioconjugation reaction types and require the incorporation of functionalities onto the nucleic acid monomers [1,2,3,4,5,6]. Taking a clue from naturally occurring modifications of exocyclic amines, we observed that N4methylcytidine (m4C) and N6-methyladenosine (m6A) occur in Watson–Crick helices of both DNA and RNA [7,8,9,10,11], where presumably the methyl group would be rotated toward the Hoogsteen edge/‘CH’-edge, leaving the second hydrogen on the Watson–Crick free to interaction in hydrogen bonding.
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