A strongly pairing fifth base: oligonucleotides with a C-nucleoside replacing thymidine

Abstract

There are five canonical bases in DNA and RNA. Each base has its particular molecular recognition properties and base pairing strength. Thymine and uracil form only two hydrogen bonds when pairing with adenine, and duplexes rich in A:T base pairs are more labile than duplexes rich in C and G, making some sequences difficult to detect via hybridization in a genomic context. Here we report the synthesis of an ethynylmethylpyridone C-nucleoside, abbreviated 'W', that presents a similar recognition surface as thymidine in the major groove but pairs with A about as strongly as C pairs with G. A phosphoramidite building block was synthesized that allows for incorporation of W residues via automated synthesis in high yield. Melting point increases over duplexes containing T:A pairs of up to 17.5°C, or up to 5.8°C per residue were measured for oligonucleotides containing W. Further, the new base shows excellent fidelity, with a single mismatched G opposite W causing a melting point depression of up to 20.5°C. The strongly pairing replacement for thymidine is only slightly larger than its natural counterpart and performs well in different sequence contexts. It can be used to target weakly pairing A-rich sequences in biological studies.

Figure 1.

Literature-known thymidine analogues and their effect on duplex stability compared to thymidine.

Figure 2.

Structure of the W residue in a DNA strand and its base pair with A in a duplex. The hydrogen at position 2 of adenine is shown to highlight the spatial proximity to the ethynyl substituent.

Scheme 1.

Synthesis of C-nucleoside 10 and its phosphoramidite building block 13. (a) Piv-Cl, NEt₃, CH₃CN; (b) H₂, Pd/C, MeOH, 2.5 bar; (c) NBS, CH₃CN, 0°C, 74% over three steps; (d) BF₃⋅OEt₂, tBuNO₂, THF, 0°C, 94%; (e) KI, CH₃CN, 76%; (f) 7, Pd(OAc)₂, P(PhF₅)₃, Ag₂CO₃, CH₃CN; (g) 3 HF⋅NEt₃, THF; (h) NaBH(OAc)₃, CH₃CN/AcOH, 0°C, 68% over 3 steps; (i) triisopropylsilylacetylene, [Pd(PPh₃)₂Cl₂], CuI, NEt₃, DMF, 80°C, 90%; (j) DMT-Cl, pyridine, DMAP, 77%; (k) (iPr₂N)₂P(OC₂H₄CN), DIPAT, CH₃CN, 88%, DIPAT = diisopropylammonium tetrazolide, DMT = 4,4′-dimethoxytrityl, NBS = N-bromosuccinimide, Piv = pivaloyl, TBAF = tetrabutylammoniumfluoride, TBDMS = tertbutyldimethylsilyl, TIPS = triisopropylsilyl.

Figure 3.

Oligonucleotides synthesized using phosphoramidite 13.

Figure 4.

HPLC chromatogram of crude 19 (C18 column, TEAA buffer/CH₃CN, 55°C), and MALDI-TOF mass spectrum of the fraction containing the oligonucleotide (inset).

Figure 5.

Representative UV-melting curves of W-containing duplexes and control duplexes of unmodified DNA, detected at 260 nm; (A) duplexes of 19 (filled circles) and 21 (open circles) with target strand 20; (B) duplexes of 18 (filled circles) and 44 (open circles) with target strand 43. The legend states which nucleobase is found in the probe strand of the respective duplex. Conditions: oligonucleotide concentration 1.6 μм (19/20/21) or 4.9 μм (18/43/44), 10 mM PIPES, pH 7.0, 100 mM NaCl, 10 mM MgCl₂. See Tables 1 and 3 for more details.

Figure 6.

Gaining insights into the structural basis for the pairing and stacking properties of W. (A) Calculated structures of ethynylmethylpyridine W and thymine (T), optimized at the B3LYP/def2-SVP-level with the molecular electrostatic potential (MEP) mapped onto the 0.001 electron density isosurface; (B) Shape complementarity between W and A, as visualized by graphically placing the two bases into close proximity. (C) Structural model explaining the suppression of wobble base pairing: While T and G can forma a wobble pair without steric conflicts, the ethynyl group of W produces a steric clash with G, and the O4 atom is not well positioned to form a hydrogen bond. The electron density and the MEP shown in (A) and (B) were computed with TURBOMOLE and visualized using TmoleX Version 4.3.1 (44).The color code ranges from –0.05 au (red) to +0.05 au (blue).