Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. Yet, the systematic mapping of the respective interactions has only started recently 1 and the main underlying mechanism proposed is chemical transformation of drugs by microbes (biotransformation). Here, we investigated the depletion of 15 structurally diverse drugs by 25 representative gut bacterial strains. This revealed 70 bacteria-drug interactions, 29 of which had not been reported before. Over half of the new interactions can be ascribed to bioaccumulation, that is bacteria storing the drug intracellularly without chemically modifying it, and in most cases without their growth being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using clickchemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the community composition through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioral response of Caenorhabditis elegans to duloxetine. Taken together, bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, likely in an individual manner.Therapeutic drugs can have a strong impact on the gut microbiome and vice versa 2-5 . The underlying drug-bacteria interactions can reduce microbial fitness 6 or alter the drug availability through biotransformation 7-14 . The latter can have either a positive or a negative impact on drug activity and efficacy. While drugs like lovastatin and sulfasalazine are chemically transformed by gut bacteria into their active forms, bacterial metabolism can inactivate drugs such as digoxin 15,16 , or cause toxic effects as in the case of irinotecan 17 .Furthering the diversity of susceptible drugs, over one hundred molecules were recently reported to be chemically modified by gut bacteria 1 . Yet, the mechanistic view on these interactions is largely confined to drug biotransformation 12,13 . Drug accumulation without metabolizationTo expand the knowledge of bacterial effect on drug availability, we systematically profiled interactions between 15 human-targeted drugs and 25 representative human gut bacterial strains (21 species; with additional subspecies or conspecific strains of Bifidobacterium longum, Escherichia coli and Bacteroides uniformis) (Supplementary Table 1). The bacterial species were selected to cover a broad phylogenetic and metabolic diversity representative of the healthy microbiota 18 (Extended Data Fig. 1a, Supplementary Table 1). On the drug side, 12 orally administered small molecule drugs (MW<500 Da), amenable to UPLC-UV-based quantificat...

A surrogate nucleobase based on 4,4'-cyanine provided up to 195-fold enhancement of red fluorescence upon complementary RNA binding. A mixture of FIT probes allowed the localization of oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by wash-free FISH and STED.

Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe-target duplex, quenching caused by torsional twisting and dye-dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (F/F0 = 23, brightness = 43 mL mol(-1) cm(-1) at λex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of oskar mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification.

Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.