Christian Grünewald

2-(1-Ethynylpyrene)-Adenosine as a Folding Probe for RNA—Pyrene in or out

A series of short RNA duplexes containing one or two 1‐ethynylpyrene‐modified adenine bases was synthesised. The melting behaviour of these duplexes was examined by monitoring temperature‐dependent pyrene fluorescence. In the singly modified RNA duplexes, the bases flanking the ethynylpyrene‐rA were varied to examine the sequence specificity of the fluorescence change of pyrene upon RNA hybridisation. Because an increase in pyrene fluorescence upon melting of the duplex can be correlated with intercalation of pyrene, and a decrease is usually associated with the position of pyrene outside the strand, a relationship between the flanking bases and the tendency of the dye to intercalate has been established. It was found that pyrene intercalation is less likely to take place if the modified base is flanked only by A–U base pairs. Flanking G–C base pairs, even only in the 5′‐direction of the modified base, will favour intercalation. In addition, we examined a doubly modified compound that had a pyrene located on each strand. The spectra indicated that the two pyrenes were close enough for interaction. Upon melting of the strand, a fluorescence blue shift corresponding to the dissociation of the pyrene–pyrene complex could be observed in addition to the intensity effect already known from the singly modified compounds. Two melting curves based on the different properties of the fluorophore could be extracted, leading to different melting points corresponding to the global duplex melting and to the change of local pyrene environment, respectively.

Ultrafast Dynamics of 1-Ethynylpyrene-Modified RNA: A Photophysical Probe of Intercalation

The photophysics of pyrene attached to an adenine base within RNA single strands and duplexes is examined with respect to the position of the pyrene within the strand and the number of pyrenes attached to one duplex. Compounds with pyrenes intercalating sequence specifically are examined, as well as a doubly modified compound, where the two pyrenes are located close enough to each other for significant excimer interaction. Femtosecond transient absorption measurements and time correlated single photon counting measurements allow a thorough examination of the local influences on the pyrene photophysics. Our results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties and lifetimes that are coupled only via the excited state as a gateway. One of them is a neutral pyrene-adenine excited state, S*, while the second one is connected to an excited charge transfer state, S*(CT). In all compounds, an ultrafast sub-ps decay from a higher excited state into the lowest excited state S* occurs, and an excited charge transfer species S*(CT) is formed within picoseconds. The fluorescence behavior of the pyrene-modified adenine, however, is strongly dependent on RNA conformation. Both S* and S*(CT) states are fluorescent, and decay within hundreds of picoseconds and approximately 2 ns, respectively. The ratio between S* and S*(CT) fluorescence depends strongly on pyrene intercalation, and it is found that the S* state is quenched selectively upon intercalation of the pyrene into RNA. The doubly modified duplex exhibits an additional fluorescent state with a lifetime of 18.7 ns, which is associated with the pyrene excimer state. This state coexists with a significant population of the pyrene monomer, since the characteristic features of the latter can still be observed. Formation of the excimer occurs on femtosecond time scales. The pyrene label thus provides a sensitive tool to monitor the local structural dynamics of RNA with the chromophore acting as a molecular beacon.

The three possible 2‐(pyrenylethynyl) adenosines: rotameric energy barriers govern the photodynamics of these structural isomers

This article presents a comprehensive study of the photophysics of 2-(2-pyrenylethynyl) adenosine and 2-(4-pyrenylethynyl) adenosine, which are structural isomers of the well-established fluorescent RNA label 2-(1-pyrenylethynyl) adenosine. We performed steady-state and ultrafast transient absorption spectroscopy studies along with time-resolved fluorescence emission experiments in different solvents to work out the interplay of locally excited and charge-transfer states. We found the ultrafast photodynamics to be crucial for the fluorescence decay behavior, which extends up to tens of nanoseconds and is partially multi-exponential. These features in the ultrafast dynamics are indicative of the rotational energy barriers in the first excited state.

Site-Directed Spin Labeling of RNA for Distance Measurements by EPR

Electron paramagnetic resonance (EPR) is a structural method and can in addition to X-ray, nuclear magnetic resonance (NMR), and FRET elucidate the structure of different macromolecular systems and determine local surroundings of paramagnetic centers in DNA and RNA. This technique permits structural characterization as well as dynamic structural changes of the macromolecular systems. In order to do so free radicals with good stability have to be introduced, most prominently nitroxide radicals. Here, we describe the site-directed spin labeling (SDSL) of DNA and RNA based on the Sonogashira cross-coupling reaction and compare to other methods available. In our method the appropriate building blocks, either 5-iodo-substituted pyrimidine or 2-iodopurine deoxy- or ribo-nucleoside phosphoramidites, were prepared and incorporated by solid-phase synthesis. Following this the protected oligonucleotides were “on column” reacted with the acetylenic nitroxide spin labels and subsequently purified. As a result the so-called “RNA ruler” for distance measurements of double-stranded RNA was developed. We present applications of this technique for DNA duplexes, DNA/RNA hybrids, RNA hairpins, riboswitches, and aptamers in vitro and “in cell.”

Synthesis of Pyrene Labeled Rna for Fluorescence Measurements

The fluorophores 1-ethynylpyrene and 1-(p-ethynyl-phenylethynyl)-pyrene were attached to RNA through a Sonogashira cross-coupling with 5-iodocytidine either in solution through phosphoamidite synthesis or via on-column conjugation during solid-phase oligonucleotide synthesis. Six probes with the sequence 5′-CUU UUC UUU CUU-3′ were derivatized with both fluorophores, whereby the position of the modified cytidine was varied. Fluorescence measurements showed sensitivity of the pyrene group to its environment in the single strands and corresponding duplexes.

Three-State Fluorescence of a 2-Functionalized Pyrene-Based RNA Label

The pyrene-based RNA-fluorescence label 2-(2-pyrenylethynyl) adenosine (2PyA) shows triexponential fluorescence, which depends strongly on the excitation wavelength. Most strikingly, a structured, long-lived fluorescence is observed in solution at room temperature after excitation into the S2 state, which is shifted hypsochromically by 30 nm compared to excitation into the S1 state. This very unusual behavior is investigated in detail with steady-state and time-resolved emission spectroscopy, ultrafast transient absorption spectroscopy, and quantum chemical calculations with both wave functions (CC2-level) and density-functional theory (DFT). 2PyA is found to emit simultaneously from two different intramolecular charge transfer states (mesomeric and twisted, MICT and TICT) which are populated most efficiently via the S1 state and a pyrene-like locally excited (LE) state. Rotational momentum derived from excess excitation energy is required to populate twisted LE configurations. Therefore, the LE state is most efficiently accessible via excitation to the S2. The stabilization of the different substates is related to two distinct reaction coordinates: the adenine-pyrene distance and the adenine-pyrene tilt angle, respectively.