Additional Base‐Pair Formation in DNA Duplexes by a Double‐Headed Nucleotide

Abstract

We have designed and synthesised a double-headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson-Crick base pairs with two complementary adenosines in a Watson-Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double-headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double-headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B-type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double-headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required.

The Extension of a DNA Double Helix by an Additional Watson–Crick Base Pair on the Same Backbone

“…The predictable formation of selective Watson–Crick base pairs and the easy introduction of chemically modified nucleotide building blocks have inspired the design of therapeutically important nucleic acids1, 2 and functional nanostructures 3. 4 A series of “double‐headed” nucleotides (nucleotides with additional nucleobases) has been presented by us5–9 and others,10–12 with the purpose of exploiting the base‐pairing or stacking interactions with the involvement of an additional nucleobase.…”

“…Recently, we reported a 2′‐deoxyuridine analogue with an additional thymine at the 2′‐position attached by a methylene linker ( U T , Scheme ) 9. Monomer U T behaves as a compressed dinucleotide, and both nucleobases participate in Watson–Crick base pairing with adenines on the complementary strand in the centre of a DNA duplex.…”

“…Monomer U T behaves as a compressed dinucleotide, and both nucleobases participate in Watson–Crick base pairing with adenines on the complementary strand in the centre of a DNA duplex. The duplex retains its B‐type structure, and the DNA backbone absorbs the discrepancy in backbone length caused by the compressed dinucleotide, thereby resulting in only local unwinding of the duplex 9. Furthermore, a surprisingly stable duplex was observed when U T monomers were placed in opposite strands (5′‐ U T A:3′A U T motif) in the so‐called (+1) zipper arrangement 9.…”

“…The duplex retains its B‐type structure, and the DNA backbone absorbs the discrepancy in backbone length caused by the compressed dinucleotide, thereby resulting in only local unwinding of the duplex 9. Furthermore, a surprisingly stable duplex was observed when U T monomers were placed in opposite strands (5′‐ U T A:3′A U T motif) in the so‐called (+1) zipper arrangement 9. This suggested the formation of a T:T base pair by the additional thymines.…”

“…This suggested the formation of a T:T base pair by the additional thymines. When compared with the unmodified 5′‐UTA:3′‐ATU motif13 (conventional T:T mismatch), the 5′‐ U T A:3′‐A U T motif exhibited an increase of around 11 °C in duplex stability 9. In the present study, we examined the double‐headed nucleotide monomer U A , which has an additional adenine introduced at the 2′ position of 2′‐deoxyuridine (Scheme ).…”