Chemical interrogation of mismatches in DNA-DNA and DNA-RNA duplexes under nonstringent conditions by selective 2'-amine acylation.
2'-Amine-substituted nucleotides in hybridized duplexes can be chemically tagged in an acylation reaction that is faster for mismatched or flexible nucleotides than for residues constrained by base pairing. Here we explore mismatch and hybridization detection using probe oligodeoxynucleotides containing single 2'-aminocytidine or -uridine nucleotides annealed to DNA or RNA targets under nonstringent conditions, below T(m). Consistent with a mechanism in which 2'-amine acylation is gated by local nucleotide flexibility, we find that efficient acylation is correlated with formation of weaker or fewer hydrogen bonds in base pair mismatches. Using 2'-aminocytidine-containing probes annealed to both DNA and RNA targets, mismatches are reliably detected as rapid selective acylation of the 2'-amine group in two sequence contexts. For probe oligonucleotides containing 2'-aminouridine residues, good discrimination between U-A base pairs and U-G mismatches could be obtained for DNA-DNA but not for DNA-RNA duplexes upon the introduction of a single 2'-O-Me group 5' to the 2'-amino nucleotide. The 2'-O-Me group introduces a structural perturbation, presumably to a more A-form-like structure, that exaggerates local flexibility at mismatches in DNA strands. Thus, 2'-amine acylation can be used to interrogate all possible mismatches in DNA-DNA duplexes and mismatches involving 2'-amine-substituted cytidine nucleotides in DNA-RNA heteroduplexes. Applications of this chemistry include detecting and chemically proofreading single nucleotide polymorphisms in both DNA and RNA targets and quantifying absolute amounts of RNA.
John
Deborah M
DM
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA.
Weeks
Kevin M
KM
D000215:Acylation; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D009692:Nucleic Acid Heteroduplexes; D015345:Oligonucleotide Probes; D012313:RNA; D004247:DNA