Stabilizing or Destabilizing Oligodeoxynucleotide Duplexes Containing Single 2′‐Deoxyuridine Residues with 5‐Alkynyl Substituents

Abstract

The 5-position of pyrimidines in DNA duplexes offers a site for introducing alkynyl substituents that protrude into the major groove and thus do not sterically interfere with helix formation. Substituents introduced at the 5-position of the deoxyuridine residue of dU:dA base pairs may stabilize duplexes and reinforce helices weakened by a low G/C content, which would otherwise lead to false negative results in DNA chip experiments. Here we report on a method for preparing oligonucleotides with a 5-alkynyl substituent at a 2'-deoxyuridine residue by on-support Sonogashira coupling involving the fully assembled oligonucleotide. A total of 25 oligonucleotides with 5-alkynyl substituents were prepared. The substituents either decrease the UV melting point of the duplex with the complementary strand or increase it by up to 7.1 degrees C, compared with that of the unmodified control duplex. The most duplex-stabilizing substituent, a pyrenylbutyramidopropyne moiety, is likely to intercalate but does not prevent sequence-specific base pairing of the modified deoxyuridine residue or the neighboring nucleotides. It also increases the signal for a target strand when employed on a small oligonucleotide microarray. The ability to tune the melting point of a DNA dodecamer duplex with a single side chain over a temperature range of >11 degrees C may prove useful when developing DNA sequences for biomedical applications.

Isostable DNA

“…62 When selecting replacements for thymidine residues, we drew on the results of our recent combinatorial search on duplexstabilizing substituents protruding into the major groove of duplex DNA. 63 A pyrenylbutyramidopropargyl ligand attached to the 5-position of 2′-deoxyuridine residues had been identified as the substituent giving the largest melting point increase. Thus far, the deoxyuridine residues carrying the substituent had to be coupled to fully assembled oligonucleotides on solid support via cross coupling of 2′-deoxy-5-iodouridine residues.…”

A 5′‐Cap for DNA Probes Binding RNA Target Strands

“…[17,23] Linker phosphoramidites (5 a-e, [8][9][10][11][12][13][14]23, and 28-30, ca. 1 mg, 2 mmol, 60 equiv) were dried together with the DNA-bearing cpg (5 mg, ca.…”

“…The modifications may involve the backbone of the probe, as in locked nucleic acids (LNAs), [9] peptide nucleic acids (PNAs), [10] phosphoramidates, [11] or oligonucleotides containing 2'-fluoronucleosides. [12] Alternatively, the nucleo-A C H T U N G T R E N N U N G bases may be modified [13,14] or substituents that aid the interrogation of the target structure may be introduced in the interior of the probes. Substituents may also be introduced at the termini of oligonucleotides.…”

5‐Substituted Nucleosides in Biochemistry and Biotechnology

“…1c), which allows the protected aminopropynyl group to be positioned at any point within a sequence [11]. The Teoc protecting group can be selectively removed with TBAF in tetrahydrofuran (THF).…”

“…The most comprehensive survey of C5 substituents based on the C5 acetylenic linker was reported by Richert and coworkers in 2004 [11]. Using the Sonogashira coupling reaction as described in Section 10.2 above, these authors prepared the oligonucleotide 5 0 -CTTTTCU Ã TTCTT-3 0 , where U Ã was one of the modified deoxyuridines shown in Scheme 10.14.…”