On-Strand Aldehyde Capture by an Indanone Building Block: A Modular Aldol Approach for Fluorescent Molecular Rotor Nucleobase Surrogates

Abstract

Fluorescent molecular rotors (FMRs) are critical tools for monitoring nucleic acid structure and function. Many valuable probes are capable of sitespecific insertion into oligonucleotides, although the methods of doing so can be arduous and lack modularity. Development of modular and more user amenable insertion methods is thus crucial to expand the biotechnological applications of oligonucleotides in what is a rapidly expanding field. Herein we report the utility of 6-hydroxy-indanone (6HI) with a glycol backbone for on-strand aldehyde capture as a modular aldol approach with synthetic ease for site-specific insertion of internal FMRs. The 6HI phosphoramidite is produced in two steps and inserted into DNA using solid-phase synthesis. On-strand aldol with aromatic aldehydes containing N-donors proceeds in quantitative yield via sodium hydroxide to create a highly functional and unique library of FMR chalcones with emissions that span the visible region (em 490- 680 nm). The FMRs provide turn-on fluorescence (Irel, 2-75-fold) upon hybridization with probe brightness up to 15,000 cm-1M-1 . The library also contains FRET pairs and dual emission probes, suitable for ratiometric sensing. The simplicity and functionality of the 6HI building block permits its future wide-spread use in a range of biosensing applications.