Glen Report 34-11: Beyond the UV Region — DEACM-dG as a Versatile Tool for Light-Activatable (“Caged”) Oligonucleotides

Authors: Janik Kaufmann and Alexander Heckel
Institute for Organic Chemistry and Chemical Biology, Goethe-University Frankfurt,
D 60438 Frankfurt, Germany
Email: [email protected]

Almost all major processes of life on the molecular and cellular scale are inherently three-dimensionally structured and time-dependent. Investigating these aspects requires tools that allow asking spatiotemporal questions. A very elegant way to do this is to use triggers so that the system under investigation can be prepared in a non-equilibrium state. After triggering, the system reacts in space and time so that we can build models about the 4D behavior. In this domain, “photocaging” has recently gained more and more interest. Technically, this process makes use of what are otherwise known as photolabile protecting groups. However, in this mode of application – as opposed to their use in organic synthesis – it is biochemical activity that is temporarily protected. Thus, light becomes the trigger signal. This is appealing because there are many different and easily available light sources (especially the vast array of high-power LEDs, currently available for low cost). There are many technologies that allow applying light with high precision (optical setups including microscopes and also catheters for an organismic application). Light is a biorthogonal trigger signal, as most cells do not respond to light. In contrast to chemical reagents, light regulation is a non-invasive method and has no cell-damaging effect as long as its wavelength lies beyond the UV region (> 380 nm). This and many more reasons make light an ideal tool for structural or kinetic investigations of biologically active compounds.


Scheme 1

Scheme 1: Simplified photolysis mechanism of DEACM with a leaving group (LG) attached to the 4-position.

Early studies only used statistic caging – a method where an oligonucleotide was incubated with unselective, electrophilic precursors of photocages to afford a randomly modified oligonucleotide with a varying number of modifications.1 However, this approach was of limited use. We2 and the groups of Deiters3 and Dmochowski4 decided to introduce photocages in specific positions – preferably the nucleobases to render them temporarily incapable of forming what is probably one of the most important features of oligonucleotides: Watson-Crick-Franklin base pairing. This approach is a very general one: on the one hand it allows the light-control of global secondary structure formation (duplex, quadruplex, stem-loop, …). On the other hand, it is also useful for regulating local, specific interactions such as for example, ligand binding in aptamers or seed region recognition in siRNAs or miRNAs.

Some applications require the incorporation of multiple photocages, while in others one single well positioned modification can already be sufficient to show the desired effect. For example, an entire 21 nt long antimiR for the regulation of angiogenesis in living mice could be blocked by incorporation of six photocages.5 Ligand binding in an aptamer on the other hand was shown to be successfully modulated with only one single caged nucleotide at the ligand binding site.6 As another example, position-specific nucleobase caging allowed the investigation of G quadruplex folding kinetics by real-time NMR spectroscopy.7 Applications of photocaging in oligonucleotides have been reviewed in the literature extensively, and more examples can be found there.8–10

One of the earliest and most popular classes of photocages are o-nitrobenzyl (ONB) cages, with 1-(2-nitrophenyl)ethyl (NPE) as the most prominent one. Their major drawbacks are their poor photorelease quantum yields and absorption maxima in the UV region, making them arguably suitable for in vivo use. Furthermore, the photolysis of ONB cages results in the formation of an o-nitrosobenzaldehyde byproduct that can potentially react with biological material to form toxic side products.11,12

Coumarin derivatives are widely known as fluorophores and by chemical modification can have absorption maxima between 300 and 700 nm.13–15  Givens et al. discovered their potential as caging groups,16 and 7-diethylamino substitution was later shown to positively influence the uncaging quantum yields.17 More importantly, the 7-diethylamino group resulted in a large red-shift of the absorption maximum out of the UV region to 400 nm and has since then been established as a commonly used substituent for coumarins.

The uncaging mechanism of coumarin cages is well understood.18 After excitation through irradiation, the molecule undergoes heterolytic cleavage of the C-O bond at the caging site, followed by nucleophilic attack and therefore addition of a solvent molecule (Scheme 1). After irradiation of a coumarin-caged nucleobase, the native nucleobase is recovered and no reactive or toxic side products are formed.

Uncaging of 7-(diethylamino)coumarin (DEACM) can be achieved by two different processes. One-photon excitation (1PE) in the region of the absorption maximum (~400 nm) is the simplest way of uncaging. Nowadays, technical progress has led to LEDs that have enough power for efficient and fast uncaging. They offer a simple and cheap solution for most uncaging experiments. In addition to 1PE, two-photon excitation (2PE) can be stimulated with femtosecond pulsed lasers allowing the uncaging of DEACM at 780 nm within the phototherapeutic window (650–900 nm) in which organic tissue has the smallest optical density. Both possibilities were demonstrated in oligonucleotides by uncaging of a DEACM-dT moiety using a 390 nm LED for 1PE and a 780 nm laser for 2PE.19

DEACM-dG specifically has been investigated by our group several years ago.20 This exact derivative is now available from Glen Research. We could show that photocleavage of the DEACM moiety was possible within wavelengths from 365 nm up to even 470 nm and that the uncaging efficiency was 17 times higher than in a nitrophenethyl photocage control. Due to the faster cleavage and higher uncaging wavelengths, it was possible to even selectively uncage DEACM in a mixture of both cages. We later found out, that we could even further drive the uncaging wavelength for DEACM to 505 nm.21 Also, the faster uncaging of DEACM makes this candidate more suitable for kinetic studies, where the photolysis process has to be faster than the kinetic process one wants to investigate.

Since DEACM-dG is based on deoxyribose, standard coupling times can be applied using either ETT or BTT as the activator. Also, cleavage and deprotection can be carried out using standard procedures in 33% aqueous NH3 at room temperature overnight. An advantageous side-effect of the DEACM-modification is its absorption and fluorescence profile, which allows easy monitoring during oligonucleotide synthesis and purification or tracking in cells.

During all steps of sample handling including deprotection, purification, or simple pipetting, ambient light exposure should be kept to a minimum. This can for example be achieved by covering the sample with aluminum foil. However, it is not necessary to work in a darkroom. Also, the use of brown tubes can make handling of photolabile samples easier. Unwanted deprotection can be further minimized by storing all samples at −21 °C in the dark. Elevated temperatures in combination with harsh conditions, e.g. basic deprotection, can lead to accidental uncaging. Therefore, we recommend keeping temperatures moderate (under 40 °C), especially during deprotection, but also purification and further handling. We decided to put our vacuum concentrators in the cold room to prevent deprotection through heat development.

The fastest uncaging of DEACM-dG can be induced by irradiation with a 400 nm LED. The uncaging rate is dependent on the amount of oligonucleotide and the number of cages installed, optical density, and the irradiation power. As a rule of thumb, quantitative uncaging of 100 pmol of an oligonucleotide containing one single cage is well achieved within 2 min of irradiation with a 160 mW LED.20 

In conclusion, DEACM-dG offers a variety of possibilities for spatiotemporal control of oligonucleotides. Its fast uncaging properties as well as higher uncaging wavelengths than common ONB cages offer better resolution in kinetic studies and even allow wavelength-selective uncaging in mixtures of both cages with high selectivity. Together with its compatibility with standard deprotection conditions and purification protocols, DEACM-dG will offer new possibilities in structural as well as kinetic studies or gene regulation, controlled by harmless blue light.

Figure 1
Figure 1: a) Coumarin core and modified DEACM. b) Photolysis of DEACM-dG in aqueous medium. c) UV/vis spectra of dG, DEACM and DEACM-dG in 1x PBS/acetonitrile 95:5.20


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Product Information

DEACM Caged-dG-CE Phosphoramidite (10-1533)

NPOM Caged-dT-CE Phosphoramidite (10-1534)