Nathan William Luedtke

Fluorescent nucleobase analogues (FBAs) are powerful reporters of nucleic acid structure and dynamics, yet strategies for programming their functional behavior directly at the hydrogen-bonding interface of DNA remain limited. Here, we introduce a nucleobase design strategy that enables conformational programming within DNA through site-specific incorporation of a 6-amino adenine analogue (^hzA) using a nosyl-protected phosphoramidite compatible with standard solid-phase synthesis. Postsynthetic condensation with aldehydes generates a structurally diverse library of hydrazone nucleobases with tunable electronic and conformational properties at the N⁶ position. Screening identified exceptionally bright FBAs (up to 4.5 × 10⁴ M^-1 cm^-1), with visible excitation and emission maxima, and quantum yields ranging from 0.004-0.863, depending on conformation, sequence context, local environment, and metal binding. Hydrazone formation can be highly fluorogenic with >120-fold increases in brightness for a coumarin-derived system, enabling real-time monitoring of reaction progress. Kinetic and thermodynamic analyses revealed that product formation and stability are strongly gated by nucleic acid structure: single-stranded DNA (ssDNA) supports rapid formation (k_on ≈ 10 M^-1 s^-1) and highly stable products (K_eq ≈ 10⁷ M^-1), whereas duplex DNA exhibits slower association rates and greater sequence selectivity than ssDNA. Notably, an ortho-phenolic hydrazone (^CySalA) forms an intramolecular hydrogen bond to N⁷ that stabilizes an anti-conformation of the hydrazone, thereby preserving selective pairing with thymidine while gating access to alternative functional states. Correct base pairing suppresses Zn²⁺ coordination and tautomer-dependent spectral transitions of ^CySalA, whereas base pair mismatches restore conformational freedom, enabling metal ion complexation with distinct absorbance/emission signatures. Collectively, this work establishes a general hydrazone-based platform for programming functional states at hydrogen-bonding interfaces, transforming a native hydrogen-bonding face into a modular chemical handle for tuning structure, reactivity, and optical responses within duplex and single-stranded DNA.

Vinyl-modified siRNAs bearing reaction sites within imperfectly paired duplex regions undergo selective IEDDA reactions with tetrazines in live cells, attenuating silencing of reporter and endogenous genes and enabling small-molecule control of RNAi.

Lipid peroxidation generates diverse aldehydic lipids associated with oxidative stress and signaling, yet methodologies capable of their detection in live cells are currently lacking. Here, we report a fluorogenic hydrazino‐Pictet–Spengler (HIPS) probe for lipidic aldehydes “FLipA‐HIPS” that generates bright, wash‐free fluorescent reaction products in vitro and in cellulo. FLipA‐HIPS is essentially nonfluorescent but reacts with aldehydes to give an environmentally sensitive fluorophore with a higher quantum yield in SDS micelles as compared to aqueous buffer alone. FLipA‐HIPS reacts with lipidic aldehydes to give 10‐ to 25‐fold higher fluorescence intensities than reactions containing either short‐chain aliphatic or aromatic aldehydes in aqueous buffer. Confocal microscopy shows that live cells pretreated with potassium bromate or the aldehydic phospholipid “POVPC” display rapid and selective FLipA‐HIPS staining that co‐localizes with the endoplasmic reticulum (ER), consistent with the ER's known role in accumulating lipidic aldehydes. These results demonstrate that HIPS reactions can exhibit useful fluorogenic properties for aldehyde biosensing applications in live cells.

Lipid peroxidation generates diverse aldehydic lipids associated with oxidative stress and signaling, yet methodologies capable of their detection in live cells are currently lacking. Here, we report a fluorogenic hydrazino‐Pictet–Spengler (HIPS) probe for lipidic aldehydes “FLipA‐HIPS” that generates bright, wash‐free fluorescent reaction products in vitro and in cellulo. FLipA‐HIPS is essentially nonfluorescent but reacts with aldehydes to give an environmentally sensitive fluorophore with a higher quantum yield in SDS micelles as compared to aqueous buffer alone. FLipA‐HIPS reacts with lipidic aldehydes to give 10‐ to 25‐fold higher fluorescence intensities than reactions containing either short‐chain aliphatic or aromatic aldehydes in aqueous buffer. Confocal microscopy shows that live cells pretreated with potassium bromate or the aldehydic phospholipid “POVPC” display rapid and selective FLipA‐HIPS staining that co‐localizes with the endoplasmic reticulum (ER), consistent with the ER's known role in accumulating lipidic aldehydes. These results demonstrate that HIPS reactions can exhibit useful fluorogenic properties for aldehyde biosensing applications in live cells.

Fluorescent probe-based quantitative polymerase chain reaction (qPCR) is essential for DNA/RNA quantification widely used in research and clinical diagnostics. The performance of fluorogenic probes depends heavily on their design, particularly the identities of the fluorophore and quencher moieties, and the linkers used to attach them to oligonucleotides. Here we report a highly modular, three-way branched glycerol ‘X’ linker in fluorogenic TaqMan® type oligonucleotide probes for multiplexed, reverse transcription qPCR (RT-qPCR). The flexible ‘X’ linker served as an internal attachment point for various quenchers (BHQ1, BHQ2) in probes containing a variable fluorophore at the 5′ end (Flu, Hex, Cy5, Cy5.5). A four-color RT-qPCR ‘tetraplex’ assay was thereby developed for distinguishing between RNA genomes from SARS-CoV-2, influenza A, and influenza B viruses in a single reaction. The ‘X’ linker exhibited superior performance with single-molecule detection limits approaching four copies, compared to an internal arabinoside-based (ara) linker strategy, demonstrating the presence of competing processes during primer extension, one where Taq exonuclease activity cleaves the fluorogenic X probe leading to productive fluorescence, and the second where the ara probe is displaced from the PCR template without cleavage. Together these results demonstrate the importance of linker structure selection in oligonucleotides for developing highly effective fluorogenic probes for qPCR.

A new approach to fluorogenic labelling is demonstrated, where a fluorescent dye undergoes disaggregation-induced emission (DIE) during an azide–alkyne ‘‘click’’ reaction with an azide-containing biopolymer to allow ‘‘no-wash’’ fluorescence imaging.

SUMMARY: Tracking of viral replication and tissue tropism by in vivo imaging can help to unveil how live-attenuated vaccines such as the yellow fever 17D (17D) work, and likewise, to understand how adverse effects develop. Here we validate 17D-TK, a reporter virus derived from 17D that expresses herpes virus thymidine kinase (TK) that specifically converts nucleoside analogues such as Ganciclovir (GCV) to induce cell death, or difluoro-EdU (dF-EdU) for bioorthogonal labelling of infected cells by Click chemistry. 17D-TK induces a cytopathic effect in infected cell cultures, as well as mortality in intracranially inoculated mouse pups in a GCV dependent manner. Preferential phosphorylation of difluoro-EdU (dF-EdU) in 17D-TK infected cells allows to selectively stain cells that support 17D replication. Prospectively, 17D-TK can be used in combination with radiolabeled tracers for real-time detection and localization of sites of active viral replication in living animals using positron emission tomography (PET).

Biomolecular assemblies are fundamental to life and viral disease. The spatiotemporal coordination of viral replication and assembly is largely unknown. Here, we developed a dual-color click chemistry procedure for imaging adenovirus DNA (vDNA) replication in the cell nucleus. Late- but not early-replicated vDNA was packaged into virions. Early-replicated vDNA segregated from the viral replication compartment (VRC). Single object tracking, superresolution microscopy, fluorescence recovery after photobleaching, and correlative light-electron microscopy revealed a stepwise assembly program involving vDNA and capsid intermediates. Depending on replication and the scaffolding protein 52K, late-replicated vDNA with rapidly exchanging green fluorescent protein–tagged capsid linchpin protein V and incomplete virions emerged from the VRC periphery. These nanogel-like puncta exhibited restricted movements and were located with the capsid proteins hexon, VI, and virions in the nuclear periphery, suggestive of sites for virion formation. Our findings identify VRC dynamics and assembly intermediates, essential for stepwise productive adenovirus morphogenesis.