Combinatorial Nucleoside-Deletion-Scaiming Mutagenesis of Functional DNA

摘要

Artificial functional DNA molecules, such as deoxyribozymes and aptamers, find increasing applications in research areas ranging from biochemistry and molecular engineering to experimental medicine. Such functional single-stranded oligonucleotides are generally identified by in vitro selection from random-sequence DNA pools. The size of the initially randomized region is mostly arbitrarily chosen, and spans from as few as 20 nucleotides (nt) to more than 200 nt. Although DNA sequences with desired activities may initially be discovered from pools with longer random regions, short sequences are usually preferred for practical applications. Computational analyses including sequence-alignment and structure-prediction methods typically need large datasets to reliably identify functional motifs. These approaches are often limited by the absence of partial randomization and reselec-tion data for reported functional sequences. Advances in this direction are expected based on increased utilization of next-generation sequencing techniques in nucleic acid in vitro selection and screening approaches. However, for the analysis and optimization of known functional nucleic acids, an experimentally simple and reliable method for minimization and characterization of active sequences is currently not available.