Tobacco-derived pyridyloxobutyl (POB) DNA adducts are unique due to the large size and flexibility of the alkyl chain connecting the pyridyl ring to the nucleobase. Recent experimental work suggests that the O4-4-(3-pyridyl)-4-oxobut-1-yl-T (O4-POB-T) lesion can undergo both nonmutagenic (dATP) and mutagenic (dGTP) insertion by the translesion synthesis (TLS) polymerase (pol) η in human cells. Interestingly, the mutagenic rate for O4-POB-T replication is reduced compared to that for the smaller O4-methylthymine (O4-Me-T) lesion, and O4-POB-T yields a different mutagenic profile than the O2-POB-T variant (dTTP insertion). The present work uses a combination of density functional theory calculations and molecular dynamics simulations to probe the impact of the size and flexibility of O4-POB-T on pol η replication outcomes. Due to changes in the Watson–Crick binding face upon damage of canonical T, O4-POB-T does not form favorable hydrogen-bonding interactions with A. Nevertheless, dATP is positioned for insertion in the pol η active site by a water chain to the template strand, which suggests a pol η replication pathway similar to that for abasic sites. Although a favorable O4-POB-T:G mispair forms in the pol η active site and DNA duplexes, the inherent dynamical nature of O4-POB-T periodically disrupts interstrand hydrogen bonding that would otherwise facilitate dGTP insertion and stabilize damaged DNA duplexes. In addition to explaining the origin of the experimentally reported pol η outcomes associated with O4-POB-T replication, comparison to structural data for the O4-Me-T and O2-POB-T adducts highlights an emerging common pathway for the nonmutagenic replication of thymine alkylated lesions by pol η, yet underscores the broader impacts of bulky moiety size, flexibility, and position on the associated mutagenic outcomes.

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.