Extended portfolio of fluorescently labeled ATPs

We now offer fluorescently labeled 7-Propargylamino-7-Deaza-ATPs, which are highly suitable substrates for in vitro transcription mediated by T7 RNA Polymerase.

To investigate the impact of linker selection on enzymatic incorporation, we synthesized a set of 7-deaza-ATP derivatives featuring different linker types: 1) without a linker, 2) with a PEG₅ linker, and 3) with a XX linker (Figure 1A). We evaluated their enzymatic incorporation properties in T7 RNA polymerase-mediated in vitro transcription and found that derivates bearing a PEG₅ linker show the best performance in terms of labeling efficiency and product yield (Figure 1B).

Figure 1: The longer the linker the higher the labeling efficiency. A) Structural comparison of ATP-AZDye488, ATP-PEG₅-AZDye488 and ATP-XX-AZDye488; B) Enzymatic properties of fluorescent 7-Deaza-ATPs with different linkers incorporated via in vitro transcription (IVT). 1400 nt RNA probes were generated by IVT using the corresponding HighYield T7 RNA Labeling Kit components. Reaction conditions: 100 ng DNA template, increasing percentage of ATP substitution by ATP-AZDye488, ATP-PEG₅-AZDye488 and ATP-XX-AZDye488 (0 - 100 %), 30 min at 37 °C (positive control: Biotin-11-ATP). Crude in vitro reaction mix was subsequently analyzed by agarose gel electrophoresis (Visualization of labeled RNA without RNA intercalator and of total RNA with ethidium bromide (EtBr)).

Our portfolio includes AZDye™ (structural analog to Alexa Fluor®), fluorescein and cyanine (Cy3 & Cy5) labeled analogs, all bearing a PEG₅ linker for efficient enzymatic incorporation (Table 1).

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