CAREER: Nucleic Acid Mutation Detection by 2'-Amine Modification

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In this research, the PI will use a new chemical method that has been developed to detect point mutations and other defects in nucleic acid sequences. The method employs oligonucleotide probes in which one 2'-ribose position (-H or -OH in DNA or RNA, respectively) is substituted with an amine (-NH2) group. 2'-amine substituted nucleotides are specifically acylated by succinimidyl esters to form a 2'-amide product. The mutation detection method exploits an unanticipated observation by the PI that 2'-amine groups at the site of a mismatch are acylated more rapidly than amine substitutions at base paired nucleotides. Acylation is specific for mismatched 2'-amine groups due to increased local flexibility. Both the 2'-amine substituted probe oligonucleotide and the target sequence may be either DNA or RNA. The method has wide potential usefulness to query mismatches and quantify hybridization with single base specificity in nucleic acid populations. Moreover, the activated reagent is a bi-functional molecule in which the mutation-sensitive acylation chemistry is linked to an independent visualization moiety. Objectives of this Career Award are, first, to explore in a fundamental way the applications, mechanism, and limitations of this chemical sensor technology, and, thereafter, to develop a practical approach for visualizing mutations in human genes. The PI has proposed new experiments for an Advanced Biochemical Laboaratory including research based experiments and a bioinformatics module, and has established a literature based course on enzyme mechanisms.9984289WeeksIn this proposal, the PI will use a new chemical method that has been developed to detect point mutations and other defects in nucleic acid sequences. The method employs oligonucleotide probes in which one 2'-ribose position (-H or -OH in DNA or RNA, respectively) is substituted with an amine (-NH2) group. 2'-amine substituted nucleotides are specifically acylated by succinimidyl esters to form a 2'-amide product. The mutation detection method exploits an unanticipated observation by the PI that 2'-amine groups at the site of a mismatch are acylated more rapidly than amine substitutions at base paired nucleotides. Acylation is specific for mismatched 2'-amine groups due to increased local flexibility. Both the 2'-amine substituted probe oligonucleotide and the target sequence may be either DNA or RNA. The method has wide potential usefulness to query mismatches and quantify hybridization with single base specificity in nucleic acid populations. Moreover, the activated reagent is a bi-functional molecule in which the mutation-sensitive acylation chemistry is linked to an independent visualization moiety. Objectives of this Career Award are, first, to explore in a fundamental way the applications, mechanism, and limitations of this chemical sensor technology, and, thereafter, to develop a practical approach for visualizing mutations in human genes. Detailed plans are provided for new courses, and creative ideas are proposed to stimulate and challenge students.