Excited-State Dynamics in the RNA Nucleotide Uridine 5′-Monophosphate Investigated Using Femtosecond Broadband Transient Absorption Spectroscopy

Brister, Matthew M.; Crespo‐Hernández, Carlos E.

doi:10.1021/acs.jpclett.9b00492

Cited by 43 publications

(56 citation statements)

References 46 publications

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“…The cross-linking experiments were performed by using a Ti:Sapphire regenerative amplifier laser system (Libra-HE, Coherent, Inc.; λ = 800 nm (center wavelength, nominal), pulse width ≤100 fs (Full Width at Half Maximum), 4.0 W at 1 kHz, contrast ratio > 1000:1 pre-pulse; > 100:1 post-pulse; root mean square (8 h) energy stability under stable environmental conditions after system warmup < 0.5 %). The 800 nm fundamental beam was converted to the 270 nm excitation beam by second harmonic sum frequency generation with an optical parametric amplifier (TOPAS, Quantronix/Light Conversion) 36 , 37 . Contributions to the excitation beam from other wavelengths were removed by a set of dichroic mirrors (λ-filter) and a Glan-Taylor polarizer 37 .…”

Section: Methodsmentioning

confidence: 99%

The kinetic landscape of an RNA-binding protein in cells

Sharma

Zagore

Brister

et al. 2021

Nature

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Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA binding proteins (RBPs) and tens of thousands of RNAs 1 . The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions 2 . However, these kinetic parameters were experimentally inaccessible in cells. Here we show that time-resolved RNA-protein crosslinking with a pulsed femtosecond UV laser, followed by immunoprecipitation and high throughput sequencing allows the determination of binding and dissociation kinetics of the RBP Dazl for thousands of individual RNA binding sites in cells. This kinetic crosslinking and immunoprecipitation (KIN-CLIP) approach reveals that Dazl resides at individual binding sites only seconds or shorter, while the sites remain Dazl-free markedly longer. The data further indicate that Dazl binds to many RNAs in clusters of multiple proximal sites. The impact of Dazl on mRNA levels and ribosome association correlates with the cumulative probability of Dazl binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects Dazl-RNA binding to Dazl function. Our results show how kinetic parameters for RNA-protein interactions in cells can be measured and how these data quantitatively link RBP-RNA binding to cellular RBP function.

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Section: Methodsmentioning

confidence: 99%

The kinetic landscape of an RNA-binding protein in cells

Sharma

Zagore

Brister

et al. 2021

Nature

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“…The experimental setup and data analysis used for the femtosecond broadband transient absorption spectroscopy (TAS) technique have been described in great deal elsewhere. [40][41][42] Briefly, the TAS spectrometer (Helios, Ultrafast Systems) make use of a Ti:Sapphire oscillator (Vitesse, Coherent), which seeds a regenerative amplifier (Libra-HE, Coherent) producing 100 fs pulses, centered at 800 nm, and with a 1 kHz repetition rate. Generation of the 267 nm excitation pulse was done as described previously, 7 by pumping an optical rail kit (FKE series, EKSMA optics).…”

Section: Transient Absorption Spectroscopymentioning

confidence: 99%

Excited State Dynamics of 7-Deazaguanosine and Guanosine 5’ Monophosphate

Krul¹,

Hoehn²,

Feierabend³

et al. 2021

Preprint

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Minor structural modifications to the DNA and RNA nucleobases have a significant effect on their excited state dynamics and electronic relaxation pathways. In this study, the excited state dynamics of 7-deazaguanosine and guanosine 5’-monophosphate are investigated in aqueous and in a mixture of methanol and water using femtosecond broadband transient absorption spectroscopy following excitation at 267 nm. The transient spectra are collected using photon densities that ensure no parasitic multiphoton-induced signal from solvated electrons. The data can be fit satisfactorily using a two- or three-component kinetic model. By analyzing the results from steady-state, time-resolved, computational calculations, and the methanol-water mixture, the following general relaxation mechanism is proposed for both molecules, Lb ® La ® 1ps*(ICT) ® S0, where the 1ps*(ICT) stands for an intramolecular charge transfer excited singlet state with significant ps* character. In general, longer lifetimes for internal conversion are obtained for 7-deazaguanosine compared to guanosine 5’-monophosphate. Internal conversion of the 1ps*(ICT) state to the ground state occurs on a similar time scale of a few picoseconds in both molecules. Collectively, the results demonstrate that substitution of a single nitrogen for a methine (C-H) group at position seven of the guanine moiety stabilizes the 1pp* Lb and La states and alter the topology of their potential energy surfaces in such a way that the relaxation dynamics in 7-deazaguanosine are slowed down compared to those in guanosine 5’-monophosphate but not for the internal conversion of 1ps*(ICT) state to the ground state.

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“…The setup is described in detail in previous publications. [48][49][50] Briefly, a Ti:Spapphire oscillator (Vitesse, Coherent, Santa Clara, CA, USA) seed a regenerative amplifier (Coherent Libra-HE) that generates a 100 fs pulses at 800 nm with a repetition rate of 1 kHz is used to pump a Traveling Optical Parametric Amplifier of Superfluorescence (TOPAS, Quantronix/Light Conversion, Vilnius, Lithuania). In this work, the TOPAS was tuned to 325 and 385 nm.…”

Section: Broadband Transient Absorption Spectroscopymentioning

confidence: 99%

Thionation of a Fluorescent Thieno[3,4-d]pyrimidine Derivative for the Development of a Heavy-Atom-Free Photosensitizer

Ortiz‐Rodríguez¹,

Fang²,

Niogret³

et al. 2021

Preprint

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All-organic, heavy-atom-free photosensitizers based on thionation of nucleobases are receiving increased attention because they are easy to make, noncytotoxic, work both in the presence and absence of molecular oxygen and can be readily incorporated into DNA and RNA. In this contribution, the DNA and RNA fluorescent probe, thieno[3,4-d]pyrimidin-4(1H)-one, has been thionated to develop thieno[3,4-d]pyrimidin-4(1H)-thione, which is nonfluorescent and absorbs near-visible radiation with about 60% higher efficiency. Steady-state absorption and emission spectra are combined with transient absorption spectroscopy and CASPT2 calculations to delineate the electronic relaxation mechanisms of both pyrimidine derivatives in aqueous and acetonitrile solutions and to explain the origin of the remarkable fluorescence quenching in the thionated compound. It is demonstrated that thieno[3,4-d]pyrimidin-4(1H)-thione efficiently populates the long-lived and reactive triplet state in hundreds of femtoseconds independent of solvent. Conversely, fluorescence emission in thieno[3,4-d]pyrimidin-4(1H)-one is highly sensitive to solvent, with an order of magnitude decrease in fluorescence yield in going from aqueous to acetonitrile solution. Collectively, the experimental and computational results demonstrate that thieno[3,4-d]pyrimidine-4(1H)-thione stands out as the most promising thiopyrimidine photosensitizer developed to this date, which can be readily incorporated as a photodynamic agent into sequence-specific DNA and RNA sequences for the treatment of skin cancer cells.

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Excited-State Dynamics in the RNA Nucleotide Uridine 5′-Monophosphate Investigated Using Femtosecond Broadband Transient Absorption Spectroscopy

Cited by 43 publications

References 46 publications

The kinetic landscape of an RNA-binding protein in cells

The kinetic landscape of an RNA-binding protein in cells

Excited State Dynamics of 7-Deazaguanosine and Guanosine 5’ Monophosphate

Thionation of a Fluorescent Thieno[3,4-d]pyrimidine Derivative for the Development of a Heavy-Atom-Free Photosensitizer

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