A family of extended 5-modified-6-aza-uridines was obtained via Suzuki coupling reactions with a common brominated precursor. Extending the conjugated-6-aza-uridines with substituted aryl rings increases the push-pull interactions yielding enhanced bathochromic shifts and solvatochromism compared to the parent nucleosides. For example, the methoxy substituted derivative 1d displays λmax abs around 375 nm, with visible emission maxima at 486 nm (Φ = 0.74) and 525 nm (Φ = 0.02) in dioxane and water, respectively.

5-(Thien-2-yl)-2'-deoxyuridine, an isomorphic fluorescent nucleoside analogue, was incorporated into multiple positions within single stranded oligodeoxynucleotides. With minimal impact on duplex stability and overall structure, oligonucleotides containing three identical isomorphic fluorescent nucleosides in alternating or neighboring positions display enhanced, sequence-dependent on-signals for either duplex formation or dissociation.

Adenosine deaminase, a major enzyme involved in purine metabolism, converts an isomorphic fluorescent analogue of adenosine (thA) to an isomorphic inosine analogue (thI), which possesses distinct spectral features, allowing one to monitor the enzyme-catalyzed reaction and its inhibition in real time. The utility of this sensitive fluorescently-monitored transformation for the high throughput detection and analysis of ADA inhibitors is demonstrated.

Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the 2P excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both thiophene-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications.

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.