Intermolecular 'cross-torque': the N4-cytosine propargyl residue is rotated to the 'CH'-edge as a result of Watson-Crick interaction

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.

Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors

“…We were thus wondering, in what way the N4-methyl group in 4mC may influence the binding of RVD HD, and if it could distinguish between 4mC and 5mC for selective analytical applications. Generally, 4mC in DNA duplexes hybridizes selectively with guanine, forming a base pair with only slightly reduced stability [44], which is also observed for larger alkyl substituents [45]. In line with these results, the N4-methyl group has been found in a Z-DNA crystal structure to be oriented towards the DNA major groove almost in plane with the pyrimidine ring to enable standard Watson-Crick base pairing [33] ( figure 1d).…”

Design and Application of Mini‐libraries of miRNA Probes for an Efficient and Versatile miRNA‐mRNA Cross‐linking

“…However, for duplex D 4 comprising the probe with the EG 0 linker (ORN‐ 4a ), the T M value (62.1+/‐ 0.3 °C) was lower than that of the unmodified parent duplex D 3 by 15 °C. Such a significant drop implied that the base‐pairing at this position was inhibited [22] . Previous studies describe that mono‐substitution on the exocyclic N 4 ‐amine of (deoxy)cytidine does not abolish Watson‐Crick base‐pairing in DNA and RNA helices, [17,20a,22,23] if the appended residue protrudes away from the Watson‐Crick face, towards the major groove of a duplex [20a,24] .…”

“…Such a significant drop implied that the base‐pairing at this position was inhibited. [22] Previous studies describe that mono‐substitution on the exocyclic N 4 ‐amine of (deoxy)cytidine does not abolish Watson‐Crick base‐pairing in DNA and RNA helices,[ 17 , 20a , 22 , 23 ] if the appended residue protrudes away from the Watson‐Crick face, towards the major groove of a duplex. [ 20a , 24 ] In the case of ORN‐ 4a , the linker was possibly too short so that the trioxsalen residue disturbed the local duplex structure.…”

Alkyne-Functionalized Coumarin Compound for Analytic and Preparative 4-Thiouridine Labeling

“…Relevant examples for site-specific cosynthetic labeling are typically found in applications of solid-phase synthesis with modified building blocks . This strategy has enabled the site-specific incorporation of dyes, non-natural nucleosides, , or bioidentical RNA modifications . In contrast, co-transcriptional functionalization of RNA with chemically modified nucleosides is distributive, thus leading to statistical incorporation of the label.…”