SELECTION OF CELL-INTERNALIZING CIRCULAR DNA APTAMERS

Abstract

<p>Adaptation of nucleic acid <em>in vitro</em> selection for whole cell targets has been demonstrated to be an effective means of isolating useful sequences with applications in biomarker detection and therapeutics. The problem of efficient delivery of materials across cell membranes is common to a variety of research and medical fields. Existing aptamers isolated in surfacing binding selections have been successfully adapted for cell targeted therapies through complex modifications. However, better aptamers may be derived from a selection optimized to isolate internalized sequences directly. A cell selection experiment with the goal of identifying circular random-sequence DNA aptamers with the ability to facilitate their own internalization into MCF7 cells was conducted. Several classes of sequences isolated from this selection were shown to target cell nuclei at a rate significantly greater than control sequences as determined by qPCR relative recovery assays supported by <em>in situ</em> RCA fluorescence microscopy data. The localization of functional DNA sequences at the subcellular and intercellular levels suggests a receptor mediated mechanism. Techniques for the selection, purification and fluorescent detection of small circular DNAs were also developed for this study. Further work to characterize and identify targets should be pursued to better understand the mechanism of internalization and judge the suitability of G18d sequences as a delivery platform.</p>