Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a ...versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.
S‐Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM‐dependent enzymes of great interest in ...biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S‐adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one‐pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin‐dependent radical SAM MT using S‐adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.
The biomimetic regeneration system for S‐adenosylmethionine (SAM) and SAM analogues presented is based on the salvage of the adenine moiety and in situ supply of d‐ribose and polyphosphate. It is compatible with a broad range of SAM‐dependent enzymes including aminopropyl transferases, and is shown to support ethylation reactions with both conventional and radical SAM methyltransferases.
The measurement of the direction of WIMP-induced nuclear recoils is a compelling but technologically challenging strategy to provide an unambiguous signature of the detection of Galactic dark matter. ...Most directional detectors aim to reconstruct the dark-matter-induced nuclear recoil tracks, either in gas or solid targets. The main challenge with directional detection is the need for high spatial resolution over large volumes, which puts strong requirements on the readout technologies. In this paper we review the various detector readout technologies used by directional detectors. In particular, we summarize the challenges, advantages and drawbacks of each approach, and discuss future prospects for these technologies.
Direct Dark Matter searches are nowadays one of the most fervid research topics with many experimental efforts devoted to the search for nuclear recoils induced by the scattering of Weakly ...Interactive Massive Particles (WIMPs). Detectors able to reconstruct the direction of the nucleus recoiling against the scattering WIMP are opening a new frontier to possibly extend Dark Matter searches beyond the neutrino background. Exploiting directionality would also prove the galactic origin of Dark Matter with an unambiguous signal-to-background separation. Indeed, the angular distribution of recoiled nuclei is centered around the direction of the Cygnus constellation, while the background distribution is expected to be isotropic. Current directional experiments are based on gas TPC whose sensitivity is limited by the small achievable detector mass. In this paper we present the discovery potential of a directional experiment based on the use of a solid target made of newly developed nuclear emulsions and of optical read-out systems reaching unprecedented nanometric resolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Direct Dark Matter searches are nowadays one of the most fervid research topics with many experimental efforts devoted to the search for nuclear recoils induced by the scattering of Weakly ...Interactive Massive Particles (WIMPs). Detectors able to reconstruct the direction of the nucleus recoiling against the scattering WIMP are opening a new frontier to possibly extend Dark Matter searches beyond the neutrino background. Exploiting directionality would also prove the galactic origin of Dark Matter with an unambiguous signal-to-background separation. Indeed, the angular distribution of recoiled nuclei is centered around the direction of the Cygnus constellation, while the background distribution is expected to be isotropic. Current directional experiments are based on gas TPC whose sensitivity is limited by the small achievable detector mass. In this paper we present the discovery potential of a directional experiment based on the use of a solid target made of newly developed nuclear emulsions and of optical read-out systems reaching unprecedented nanometric resolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We report that the measurement of the direction of WIMP-induced nuclear recoils is a compelling but technologically challenging strategy to provide an unambiguous signature of the detection of ...Galactic dark matter. Most directional detectors aim to reconstruct the dark-matter-induced nuclear recoil tracks, either in gas or solid targets. The main challenge with directional detection is the need for high spatial resolution over large volumes, which puts strong requirements on the readout technologies. In this paper we review the various detector readout technologies used by directional detectors. In particular, we summarize the challenges, advantages and drawbacks of each approach, and discuss future prospects for these technologies.
Direct Dark Matter searches are nowadays one of the most fervid research topics with many experimental efforts devoted to the search for nuclear recoils induced by the scattering of Weakly ...Interactive Massive Particles (WIMPs). Detectors able to reconstruct the direction of the nucleus recoiling against the scattering WIMP are opening a new frontier to possibly extend Dark Matter searches beyond the neutrino background. Exploiting directionality would also prove the galactic origin of Dark Matter with an unambiguous signal-to-background separation. Indeed, the angular distribution of recoiled nuclei is centered around the direction of the Cygnus constellation, while the background distribution is expected to be isotropic. Current directional experiments are based on gas TPC whose sensitivity is limited by the small achievable detector mass. In this paper we present the discovery potential of a directional experiment based on the use of a solid target made of newly developed nuclear emulsions and of optical read-out systems reaching unprecedented nanometric resolution.
Nowadays there is compelling evidence for the existence of dark matter in the Universe. A general consensus has been expressed on the need for a directional sensitive detector to confirm, with a ...complementary approach, the candidates found in conventional searches and to finally extend their sensitivity beyond the limit of neutrino-induced background. We propose here the use of a detector based on nuclear emulsions to measure the direction of WIMP-induced nuclear recoils. The production of nuclear emulsion films with nanometric grains is established. Several measurement campaigns have demonstrated the capability of detecting sub-micrometric tracks left by low energy ions in such emulsion films. Innovative analysis technologies with fully automated optical microscopes have made it possible to achieve the track reconstruction for path lengths down to one hundred nanometers and there are good prospects to further exceed this limit. The detector concept we propose foresees the use of a bulk of nuclear emulsion films surrounded by a shield from environmental radioactivity, to be placed on an equatorial telescope in order to cancel out the effect of the Earth rotation, thus keeping the detector at a fixed orientation toward the expected direction of galactic WIMPs. We report the schedule and cost estimate for a one-kilogram mass pilot experiment, aiming at delivering the first results on the time scale of six years.
The measurement of the direction of WIMP-induced nuclear recoils is a compelling but technologically challenging strategy to provide an unambiguous signature of the detection of Galactic dark matter. ...Most directional detectors aim to reconstruct the dark-matter-induced nuclear recoil tracks, either in gas or solid targets. The main challenge with directional detection is the need for high spatial resolution over large volumes, which puts strong requirements on the readout technologies. In this paper we review the various detector readout technologies used by directional detectors. In particular, we summarize the challenges, advantages and drawbacks of each approach, and discuss future prospects for these technologies.
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