In-vivo targeted tagging of RNA isolates cell specific transcriptional responses to environmental stimuli and identifies liver-to-adipose RNA transfer

J. Darr; M. Lassi; Archana Tomar; R. Gerlini; F. Scheid; M. Hrabe de Angelis; M. Witting; R. Teperino

Small molecule design and genetic approach for targeted in-vivo labeling of RNA 93 5-Ethynyl Uridine (5EU) is a synthetic uridine analogue extensively used in RNA turnover studies 94 [27] [28] [29] . The nitrogenous base contains an alkyne group that can be covalently linked to an azide 95 group using a simple copper mediated reaction called click chemistry [30, 31] . This synthetic base 96 has been demonstrated to incorporate into transcribing RNA in place of uridine and have little to 97 no biological effects thereafter [27] . Following administration to mice, 5EU is readily taken up by 98 cells with no regard to cell identity, depending to some extent on the administration method and 99 dosage used [27] . Here, we present a novel method for the targeted in-vivo delivery of 5EU. 100 To achieve this, we designed a 'pro-drug' of the 5EU base (HD5EU) that is based on the 'Hep-101 Direct' pro-drug design [32, 33] (Figure 1a) . This design was developed to target small molecules 102 and nucleotide analogues to the human CYP3A4 enzyme and several small molecules of this 103 design have been or are currently in clinical studies [34] [35] [36] . The human CYP3A4 enzyme 104 catalyzes an oxidative cleavage of the HD5EU small molecule which, following a spontaneous 105 beta-elimination, results in the formation of 5EU mono-phosphate that can then be incorporated 106 into transcribing RNA (Figure 1a ). HD5EU was synthesized by Chiroblock GmbH, the identity 107 of the final product was validated using MS, p-NMR and h-NMR and the molecule's purity was 108 assessed at over 98% (Sup. Figure 1a-d) . 109 In addition to the HD5EU small molecule we took advantage of the published humanized liver 110 specific CYP3A4 mouse line FVB/129P2-Cyp3a13 tm1Ahs Del(5Cyp3a57-Cyp3a59) 1Ahs 111 Tg(APOE-CYP3A4) A1Ahs obtained from Taconic [37, 38] . These humanized mice (hCYP3A4) 112 express the human CYP3A4 enzyme under a modified Apolipoprotein E (APOE) promoter and 113 are stably knocked out for nine homologous murine genes, thus leaving the human enzyme as the 114 sole member of the enzyme family to be expressed in a Cre-independent, tissue-specific manner 115 in-vivo. In keeping with published data on the activity of the modified ApoE promoter [39] , qRT-116 PCR and WB analyses demonstrate restricted expression of the human Cyp3a4 enzyme to liver 117 and kidney (Figure 1b-c) . As such, upon administration of the HD5EU small molecule to the 118 humanized CYP3A4 mice, we expect the molecule to be metabolized to bioavailable 5EU mono- 143 To test and validate the metabolism of the HD5EU small molecule, we first isolated primary 144 hepatocytes from hCYP3A4 mice. Following an 8 hour treatment of primary hepatocytes with 145 1mM HD5EU or 5EU, nuclear staining similar to 5EU labeling is clearly evident following click- We administered HD5EU to humanized CYP3A4 mice and already 2 hours following 153 administration found robust nuclear staining in hepatocytes and kidney epithelial cells but in no 154 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/670398 doi: bioRxiv preprint other tissue examined. This in contrast to mice administered with 5EU where nuclear staining was 155 evident in multiple tissues (Figure 2b-c and sup. Figure 2a) . Of note, animals administered with 156 HD5EU did not demonstrate any visible side effects. In additio