Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of  Computational approaches have recently made significant progress in the protein engineering field evolving from a tool for helping experimentalists to prioritize or short-list mutations for testing to being capable of making fully reliable predictions. However, not all the fields of protein modeling are evolving at a similar pace. That is why evaluating the capabilities of computational tools on different tasks is important to provide other scientists with up-to-date information on the state of the field. Here we tested the performance of Rosetta (one of the leading macromolecule modeling tools) in improving small molecule-binding proteins. We successfully redesigned a fluorogen binding protein DiB1 –a protein that binds a non-fluorescent molecule and enforces its fluorescence in the obtained complex–for improved brightness and better performance in super-resolution imaging. Our results suggest that such tasks can be already achieved without laborious library screenings. However, the flexibility of the proteins might still be underestimated during standard modeling protocols and should be closely evaluated. The biological role of many natural fluorogens is often directly connected to their ability to absorb light. For example, chlorophyll is used in photosystems of cyanobacteria, algae, and plants; flavins are essential parts of DNA photolyases [Fluorogenic molecules are compounds whose ability to fluoresce can be modulated, for example, by a chemical modification, change in the environment, or electronic structure . A numbetolyases  and cryptolyases ; rhodopstolyases .A number of fluorescent probes has been created by mutating natural fluorogen-binding proteins to promote their fluorescence. For example, starting from bacterial light-oxygen-voltage–sensing domains, such an effort yielded flavin-binding fluorescent proteins ; differeIn addition to natural fluorophores, fluorogenic compounds can be synthesized. The existing examples include but are not limited by BODIPY dyes, rhodamines, cyanines, and coumarins . The utiIn silico shortlisting with the further screening of the lipocalin Blc mutants launched DiBs FAPs tags family binding green [de novo ligand-binding β-barrel protein design using Rosetta followed by two additional rounds of modeling-directed mutagenesis of proteins selected by in vitro screening of a limited number of hits from the previous step yielded two DFHBI-binding proteins designated mFAP1 and mFAP2 [Several FAPs have been designed so far to use unnatural fluorogenic molecules. Screening of libraries of antibodies resulted in the discovery of binders for derivatives of thiazole orange, malachite green , and cyang green  and red ng green  BODIPY dng green . Finallynd mFAP2 .in silico modelling of more than 100 000 mutants followed by a rigid body docking of a library of green fluorescent protein (GFP) chromophore-like ligands. Nineteen mutants and ten fluorogens were shortlisted to be evaluated experimentally [in silico might be already possible.All FAP modeling projects described above involved screening of libraries of proteins to select the one with the desired properties, which is time and resource consuming. For example, the first-generation lipocalin Blc-based FAPs (DiBs) were obtained after mentally . HoweverHere we tested the performance of one of the leading macromolecules modeling suites, Rosetta , on the Kd [DiB1 protein was selected as a starting point for the protein design project. Among first generation Blc-based FAPs this mutant showed the lowest Kd  indicatiKd . For thiKd . The besFig) was performed with gradually decreasing ligand sampling freedom until the solution converged on a single binding pose . First, 5 000 protein-ligand complexes were generated using a coarse protein binding pocket sampling strategy with 5 Å maximum ligand translation allowed per step and up to 360° rotation. The top 500 models by total energy were further sorted based on their protein-ligand interface score and the 50 best were selected for a subsequent docking round. During the next step ligand translation and rotation were restricted to 1 Å and 45°, respectively. 100 structures were generated for each of 50 starting models resulting again in a total of 5 000 output structures. 50 best structures were selected as previously and the last round of docking was performed with 0.2 Å maximum translation and 5° maximum rotation for fine-tuning of the ligand placement. The best scoring docking pose was further used for the ligand binding pocket design. In this model the ligand was located within an interaction distance from amino acids at positions 141 and 74, mutations in which were shown to influence the properties of the ligand:protein complex in our previous study [Docking of the M739 chromophore Fig was ing pose Fig. Firus study . While tus study . TherefoFig). The protocol was previously shown to perform better on apolar small molecules whose binding is dominated by van der Waals interactions [In fluorescent proteins, their ability to fluoresce and the brightness of the fluorescence highly depend on the capacity of the surrounding amino acids to stabilize the chromophore in a planar conformation . In the case of M739, the structure is already conformationally locked in the favorable planar conformation. Contrary, its fluorescence quantum yield (QY) was shown to vary dramatically depending on the solvent with an almost tenfold increase in dioxane compared to water . Thus, hractions .From the starting complex structure, we generated 5 000 designs. Amino acids with their Cα atoms within 6 Å of any ligand atom or with their Cα atoms within 8 Å of any ligand atom and with their Cα-Cβ vector pointing toward the ligand were allowed to be designed to any amino acid except cysteine. The residues with their Cα atoms within 10 Å of any ligand atom or with their Cα atoms within 12 Å of any ligand atom and their Cα-Cβ vector pointing toward the ligand were allowed to repack. The 2 000 best scoring designs were then sorted based on their ligand-protein interface score and the best 50 were selected for detailed analysis.Fig 1A and 1B). Mutations at positions 74, 108, 109, 116, 139, and 141 were relatively rare with the majority of the generated sequences retaining the native amino acid at those positions. In contrast, amino acids at positions 53, 76, 89, 90, and 107 were mutated in almost all 50 selected models. Four of these positions showed strong convergence toward one specific mutation: Phe at position 76, Tyr at position 89, Val at position 90, and Ala at position 107. Amino acid 53 was mutated to either alanine or glutamine with nearly equal frequency in the models.A total of 16 amino acid positions were found to be mutated at least in one of the selected designed models  contained only mutations at these five positions. Four residues  were converted to the most favorable amino acids for these positions. Alanine, the second most frequent amino acid in position 53 among 50 best-scored poses, was found in position 53 in that sequence. After additional manual examination, this sequence (designated DiB-RM) was selected for testing.Of note, one of the top-scoring models (in vitro (Table 1). In comparison with DiB1, the DiB-RM:M739 complex, with almost identical fluorescence spectra, demonstrated an increase in both quantum yield (QY) and extinction coefficient (ɛ) resulting in approximately 25% increased brightness. These changes were accompanied by some loss in the apparent binding affinity of the protein to the ligand.We first compared the photophysical properties of DiB-RM in complex with M739 chromophore with the parental protein DiB1 in cellulo . In concordance with the results obtained in vitro, DiB-RM demonstrated increased brightness compared to DiB1: ~ 1.38:1 versus ~ 1.29:1 observed in vitro.We next characterized the DiB-RM protein using transiently transfected living cells. We first generated a fusion protein of DiB-RM with histone H2B and blue fluorescent protein TagBFP, which was compared with a previously created H2B-TagBFP-DiB1 construct . Using TFig 2F and 2G).We next created DiB-RM fusions with various structural proteins  and imaged transiently transfected mammalian cells using confocal fluorescence microscopy. The target intracellular structures were brightly fluorescent immediately upon the addition of the fluorogen and did not show any signs of mislocalization or aggregation  and tested the behavior of the fusion protein composed of the newly obtained N fragment (designated DiB-RM-splitN1-109) with a fluorescent protein mNeonGreen in transiently transfected HEK cells. Interestingly, we did not observe aggregation  as was seen previously for DiB2-split N fragment in a similar experiment. Inspired by this observation we also checked free DiB-RM-split C fragment distribution in cells. There is only one amino acid difference between DiB2 and DiB-RM proteins downstream to the split point (L141N) and it did not affect the performance of the DiB-RM-split C fragment: HEK cells transiently transfected with the DiB-RM-splitC110-177-TagBFP construct showed uniform labeling in the blue fluorescent channel . We then tested the self-assembly capability of the DiB-RM N and C fragments obtained via split between amino acid residues 109 and 110. Transient cotransfection of H2B-DiB-RM-splitN1-109 and TagBFP-DiB-RM-splitC110-177 constructs in living cells revealed successful attraction of TagBFP-DiB-RM-splitC110-177 fusion protein to the nuclei despite lacking a nuclear localization signal  and staining nuclei in green upon the chromophore addition  as a result of efficient self-assembly of split-DiB-RM.We previously created a self-assembling split system from the first generation of DiB proteins . Despiteregation Fig as w channel Fig. We in vitro (Table 1) using purified protein. As was previously observed for other self-assembling DiB-splits [We also characterized the DiB-RM-split:M739 complex B-splits , splittiS3 Movies). All three tags provided reconstructions of vimentin fibers with better resolution than widefield fluorescence microscopy . DiB1 showed an initial exponential decrease in the number of localizations that later turned into a linear decrease down to ~20% of the initial localization count. The DiB-RM signal decreased only linearly throughout the experiment. That allows for the accumulation of a much higher number of localizations during the same time period using DiB-RM. DiB-RM-split tag performed similarly to DiB1 . DiB-RM also demonstrated higher single-molecule brightness  than DiB-RM-split and DiB1 . Both DiB1 and DiB-RM-split provided lower localization precision  compared to DiB-RM .The main power of DiB FAPs lies in single-molecule localization microscopy of living cells ,14. We tapo form. The asymmetric unit contains two copies of the protein with the canonical lipocalin fold, a β-barrel with an α-helix . The two copies align well (0.77 Å rmsd across 158 Cα atoms) with the main difference found in the E/F loop and at the N terminus of the protein .To further characterize DiB-RM we obtained a number of crystal structures. We first successfully crystalized full-length DiB-RM in Fig 4C).Interestingly, during structure refinement, we discovered positive difference density map features in the binding pocket of the molecules. Based on the shape of the density we speculated that they belong to the dodecyl chains of n-Dodecyl-β-D-maltoside (DDM) present in the crystallization buffer . Unfortunately, we still observed the same long carbon chain-like density in the binding pocket of DiB-RM in these crystals. It might be explained by the higher affinity of DiB-RM to DDM than to M739 or by the requirement of the presence of DDM in the binding pocket of the protein for crystal formation in these conditions.We also obtained colored crystals in similar conditions using soaking or co-crystallization of DiB-RM with M739 Fig. UnfTo test the former hypothesis, we assessed the binding affinity of DDM to DiB-RM using tryptophan fluorescence quenching assay. Surprisingly, we have not detected any spectral changes upon the addition of up to 50x molar access of the DDM to the protein solution in normal buffer conditions. While this experiment cannot fully reproduce the processes and their dynamics that are happening during crystallization, the requirement of the presence of DDM for crystal formation in these conditions seems to be a more likely explanation.apo DiB-RM crystals in DDM-free conditions. This crystal also contains two protein molecules in the asymmetric unit . However, the relative orientation of the molecules differs: we observed approximately 50 degrees rotation . This observation along with the data from ’Protein interfaces, surfaces and assemblies’ service PISA [Fig 4E).We later obtained other ice PISA  suggestsFig 4F). Unsurprisingly, the crystals remained clear after the addition of the ligand in the drops.In comparison to the first structure, the main difference was observed in the conformation of the E/F loop of the protein. In the new structure, this loop is bent inwards and almost fully closes the entrance to the ligand binding pocket of the protein . Despite the backbone cleavage, the lipocalin fold is well preserved as it was previously observed for other DiB-derived split proteins [Fig 4I).We also obtained DiB-RM-split proteins  Fig). The carbon chain-like density in the binding pocket was absent, which further supports our hypothesis that the previously observed density in full-length DiB-RM crystals indeed belongs to DDM and not to some other molecule co-purified with the protein. However, we were not able to locate any density for the M739 ligand either.Multiple colored crystals from DiB-RM-split:M739 mixture were obtained in 1.8 M lithium sulfate and 6% 2-propanol buffer in presence of different additives Fig. Thein vitro (except for the dissociation constant) and as a tag for protein-PAINT [Table 2) of the DiB-RM:M739 complex compared to the parental DiB1:M739 through the better protein:ligand interface packing, in the absence of the ligand these bulky amino acids might partially hinder the entrance of the ligand in the binding pocket.Using a fixed backbone Rosetta Design protocol, we predicted a set of mutations to improve the first generation lipocalin Blc-based FAP DiB1. The resulting protein, designated DiB-RM, performs better than its parental variant both in-PAINT  with hig1-109 fragment, and the split protein successfully assembled in living cells without the need of including the additional overlapping β strand in the C fragment. Regardless of lower brightness both in vitro and in super-resolution microscopy set up as well as less stable fluorescent signal compared to full-length DiB-RM tag, we speculate that the DiB-RM-split should be a scaffold of choice for further DiB-split system optimizations.Despite no intentional optimization for it, the DiB-RM-based split protein (DiB-RM-split) created analogously to the previously tested DiB2-split system  also behapo Blc structures [Fig 5A) and served as an entry point for the vaccenic acid that was previously co-crystallized with this lipocalin [Fig 5B), the chromophore was found much deeper and angled . This became possible due to the rotation of the lipocalin E/F loop. While high flexibility of this loop was previously shown [Fig 5C).Recently, the structure of DiB1 in complex with the chromophore M739 has been solved . To our ructures ,25 Despite our inaccurate initial docking of the chromophore, it is obvious that the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the DiB tag. To further investigate this phenomenon, we ran a number of experiments.Figs S7A) confirming that steric clashes with the backbone most likely caused our wrong initial binding mode prediction.First, to test the possibility that using a starting position of the E/F loop incompatible with the binding mode observed in the crystal structure was responsible for the failure of Rosetta docking, we reran the docking using either the DiB1 crystal structure minimized in the absence of ligand, or the same DiB1 model that was generated for the initial docking but with deleted E/F loop (-6 amino acids). In both cases, all 50 top-scored structures had a crystal-like M739 placement . This observation together with our inability to obtain the DiB-RM:M739 co-crystal structure might indicate that we have stabilized an alternative binding site for the ligand by the introduced mutations but have not destroyed the other one. As a result, the ligand in DiB-RM might have multiple possible binding positions. This can also explain the larger apparent dissociation constant of this new complex.We then performed docking using the DiB1-based DiB-RM model generated the same way as the DiB1 model has been prepared before from the Table 2).To further investigate the role of the five introduced mutations we created and analyzed five reverse single mutant variants of DiB-RM , it is not surprising that this phenylalanine has been mutated by Rosetta. This substitution might be also important for the protein’s performance in the crowded cell environment.All -exposed Fig, it Kd accompanies by the bathochromic shift of the fluorescence spectra and a decrease in both QY and ɛ. The side chain of the amino acid in position 76 packs against the ligand . These spectral changes upon introduction of the polar residue next to the ligand align well with the effects of the polarity of the environment on the chromophore M739 properties observed in the free ligand model [Reintroduction of asparagine at position 76 is the only mutation that results in tighter binding to the chromophore. That might be explained by the ability of bulkier phenylalanine to sample side chain conformations that are not compatible with the ligand’s entrance to the binding pocket or its correct placement there. However, the lower e ligand Fig. Thend model .Fig). The observed changes might support the presence of an alternative ligand binding mode in DiB-RM where Y89 packs against the ligand. Alternatively, the introduction of tyrosine at position 89 can induce more complex rearrangements in the structure of the protein. For example, through interactions with the aromatic amino acids-rich flexible E/F loop.Reversing of the other introduced aromatic residue, DiB-RM-Y89S, results in an almost four-fold decrease in binding affinity as well as moderate spectral shifts and decrease in brightness similar to ones observed in DiB-RM-F76N mutant. The amino acid in position 89 is not in direct contact with the ligand, assuming DiB-RM interacts with the ligand similar to what is seen in the DiB1:M739 co-crystal structure Fig. TheFig). The most likely explanation of Rosetta’s favoring of the E90V mutation is the better β-sheet propensity of valine compared to glutamate [Kd upon reversion of this mutation might indeed indicate its effect on the overall stability of the protein. Even less pronounced in vitro changes caused by A107S substitution located in a relatively flexible region of the protein (at the base of the E/F loop) make it difficult to propose its role, if any.The remaining two variants, DiB-RM-V90E and DiB-RM-A107S, carry substitutions at the positions that are pointing outside of the ligand-binding cavity Fig. Thelutamate . The obsOverall, it appears that the majority of the mutations proposed by Rosetta were beneficial for the DiB-RM performance with the polarity of the residues in the proximity of the ligand influencing the photophysical properties of the FAP the most. Hence, refining of the protein:ligand interface using a conventional RosettaLigand design protocol seems to be a possible option for optimization of FAPs with rigid, apolar ligands. The decrease in the ligand binding affinity might be further avoided in the future by employing a multistate design protocol  with simapo form in different conditions further confirmed the already known high flexibility of the E/F lipocalin loop that was not properly addressed during our computational redesign. Rigidifying of this loop through shortening or designed interactions can provide better stabilization of the ligand in the binding pocket. This in turn can allow for expanding the compatible ligand libraries towards more flexible, conformationally unlocked chromophores [Here we explored the power and limitations of Rosetta for the redesign of a protein-ligand complex. Our work resulted in the creation of an improved FAP-based fluorescent tag, however, potentially through stabilization of an alternative ligand binding site. Future optimization of DiB-RM might be focused on disabling one of the two suggested binding sites of the ligand. Crystallization analysis of DiB-RM in the mophores ,30. SuchFive mutations suggested by computational modeling were introduced into the previously described DiB1-pBAD bacterial expression vector  by self-The DiB-RM-split vectors for bacterial protein expression were created using plasmids pMRBad-Z-CspGFP (Addgene plasmid #40730), pET11a-Z-NspGFP (Addgene plasmid #40729) , and theThe H2B-TagBFP-DiB-RM plasmid was constructed by self-assembling cloning  using DiAll other plasmids for mammalian expression were assembled using Golden Gate cloning following MoClo standard –36. EachThe resulted constructs’ amino acid sequences are provided below. The linker sequence is underlined.>vimentin-DiB1DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>vimentin-DiB-RMDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>vimentin-DiB-RM-split1-109DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>DiB-RM-split110-177-TagBFPGDPPVATMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*MGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS>mNeonGreen-DiB-RM-split1-109DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFS*MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYKThe correctness of all obtained constructs was confirmed by sequencing.E. coli strain. Cells were grown in LB media or M9 minimal media supplemented with 100 μg/mL ampicillin (full-length DiB proteins) or 100 μg/mL ampicillin and 50 μg/mL kanamycin (split protein) at 37°C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10 μM IPTG (split protein) at 0.8 OD. Cells were harvested after 3 hours of expression in LB or after overnight expression in minimal media at 37°C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at -80°C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts.All lipocalin proteins were expressed in XJb(DE3) Autolysis (Zymo Research) E. coli strain. Cells were grown in LB media supplemented with 100 μg/mL ampicillin at 37°C. Expression was induced by the addition of 500 μM IPTG at 0.8 OD. Cells were harvested after overnight expression at 18°C. Before purification cells were resuspended in PBS buffer, pH 7.4, and sonicated on ice. The lysates were centrifuged to obtain cell-free extracts.Fluorescent protein Venus was expressed in BL21(DE3) The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg column  pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.Protein concentrations were estimated using the Bradford dye-binding method-based  colorimeKd). For each protein, the measurements were performed using at least two independent protein purifications and at least three technical replicates for each protein sample.Titrations were performed and analyzed as previously described  using FlHoriba Jobin Yvon Fluoromax-3 fluorometer was used to detect full fluorescence excitation and fluorescence emission spectra for excitation/emission maxima evaluation.apo form and in the presence of ~0.5–3.0 μM of the M739 chromophore. FAP concentrations for experiments were chosen individually for each protein based on the previously calculated Kd values to ensure that at least 95% of the added chromophore is bound to the protein . Spectra of the corresponding apo FAP solutions were subtracted from the absorption spectra of the protein-fluorogen complexes. The absorption at 510 nm values from these corrected spectra was also plotted against the area under the corresponding fluorescence emission curves and the linear approximation of the correlation has been calculated. The QYs were then calculated as a ratio of the slopes of the protein of interest and standard curves multiplied by standard’s QY. For each protein, the measurements were performed using protein aliquots from at least two independent protein purifications. Reported is a mean value.Fluorescence quantum yield (QY) was measured relative to the fluorescent protein Venus . First, FAPε is the FAP-fluorogen complex extinction coefficient, FAPA–the FAP-fluorogen complex absorption at maximum, AM739–free chromophore absorption at the FAP-fluorogen complex absorption maximum, cM739–total added chromophore concentration, and α–a fraction of added chromophore that is bound to the protein calculated based on the previously determined FAP-fluorogen complex Kds. Reported is a mean value ± s.d. of measurements obtained for at least two independently expressed and purified protein samples using at least five data points per sample.Absorption spectra collected for QY calculations were also used for excitation coefficients calculations. For each of the FAP-fluorogen complexes, the complex absorption maximum has been determined. Free M739 chromophore spectra were used to define free chromophore contribution to absorption at the given wavelength and to calculated chromophore concentrations. The FAP-fluorogen complexes’ extinction coefficients were calculated using the following equation:apo DiB-RM  was crystalized at 21°C in 2 M ammonium sulphate, 2.5% 2-propanol supplemented with 5% w/v n-Dodecyl-b-D-maltoside according to the Hampton Research Additive Screen protocol using hanging drop vapor diffusion technique. Crystals grew within 1–3 days.Full-length apo DiB-RM  were also obtained at 21°C in 1.5 M lithium sulfate, 0.1 M tris hydrochloride pH 7.0, and 5% 2-propanol, supplemented with 200 mM cesium chloride using hanging drop vapor diffusion technique. For this 2 μL of protein solution were mixed with 1.2 μL of crystallization buffer  and 0.8 μL of 1 M cesium chloride from Hampton Research Additive Screen. Crystals grew within 2–3 weeks.Crystals of different morphology of full-length apo crystals were obtained at 21°C in 1.6 M ammonium sulfate, 0.1 M MES, pH 4.5 supplemented with 5% w/v n-Dodecyl-b-D-maltoside according to the Hampton Research Additive Screen protocol using hanging drop vapor diffusion technique. Crystals grew within 1 week.DiB-RM-split  All crystals were flash-frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.Table. The models have been deposited into the Protein Data Bank . Structure figures were prepared using PyMol .Diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G or 21-ID-F at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using the xia2 software suite . The cry2. For transient transfections, FuGENE HD reagent (Promega) was used. Immediately before imaging DMEM was replaced with HHBS media (Hanks Buffer (PanEco) supplemented with 20 mM HEPES (Sigma)).HEK293 and HeLa Kyoto cells were grown in Dulbecco’s modification of Eagle’s medium (DMEM) (PanEco) supplied with 50 U/ml penicillin and 50 μg/ml streptomycin (PanEco), 2 mM L-glutamine (PanEco), and 10% fetal bovine serum  at 37°C and 5% COWidefield fluorescence microscopy was performed with the Leica DMI6000B inverted microscope equipped with HC PL Apo 100x NA 1.40 oil lens and HC PL Apo 40x NA 0.85 lens, CoolLED pE-300 light source, Zyla 5.5 sCMOS camera (Andor), using GFP and BFP filter sets.Confocal imaging was performed using an inverted Leica confocal microscope DMIRE2 TCS SP2  equipped with HCX PL APO lbd.BL 63.0x NA 1.40 OIL objective, excitation by 488 nm laser line (100 μW).-2 of 488 nm laser light, 33 ms frame exposure time, for 10,000 frames.Single-molecule localization super-resolution imaging of living cells was performed with Nanoimager S  microscope at 37°C. The microscope was equipped with Olympus UPlanSApo 100x NA 1.40 oil immersion objective. Imaging experiments performed with 1.1 kW cmAll Rosetta runs were performed with weekly release 2015.12.57698.The A36C and L141N mutations were manually introduced into the crystal structure (PDB ID 1QWD). The structure was further minimized using Rosetta Relax application  with the-flip_HNQ-no_optH false-relax:constrain_relax_to_start_coords-relax:ramp_constraints false-nstruct 50-ex1-ex2-use_input_scThe M739 chromophore geometry was optimized by the density functional method RB3LYP, using the 6–311+G** basis set, a restricted hybrid HF-DFT SCF calculation was performed using Pulay DIIS + Geometric Direct Minimization to get a set of ideal bond lengths and angles. The conformers library for the ligand was further generated using BCL::Conf conformer generator .Docking of the M739 chromophore was performed in three steps. First, 5 000 structures were generated using 5 Å maximum ligand translation allowed per step and up to 360° rotation. 50 structures were selected for the next docking round during which ligand translation and rotation were restricted to 1 Å and 45° correspondingly. 100 structures were generated for each of the 50 starting models. Analogously 50 best structures from the second docking step were selected and the last round of docking was performed with 0.2 Å maximum translation and 5° maximum rotation.The following RosettaScripts protocol has been used with the appropriate changes introduced to the Transform Mover during the second and the third docking rounds:<ROSETTASCRIPTS><SCOREFXNS>    <ligand_soft_rep weights = ligand_soft_rep>        <Reweight scoretype = fa_elec weight = 0.42/>        <Reweight scoretype = hbond_bb_sc weight = 1.3/>        <Reweight scoretype = hbond_sc weight = 1.3/>        <Reweight scoretype = rama weight = 0.2/>    </ligand_soft_rep>    <hard_rep weights = ligandprime>        <Reweight scoretype = fa_intra_rep weight = 0.004/>        <Reweight scoretype = fa_elec weight = 0.42/>        <Reweight scoretype = hbond_bb_sc weight = 1.3/>        <Reweight scoretype = hbond_sc weight = 1.3/>        <Reweight scoretype = rama weight = 0.2/>    </hard_rep></SCOREFXNS><SCORINGGRIDS ligand_chain = "X" width = "16">    <vdw grid_type = "ClassicGrid" weight = "1.0"/></SCORINGGRIDS><LIGAND_AREAS>    <docking_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false minimize_ligand = 10/>    <final_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false/>    <final_backbone chain = X cutoff = 7.0 add_nbr_radius = false all_atom_mode = true Calpha_restraints = 0.3/></LIGAND_AREAS><INTERFACE_BUILDERS>    <side_chain_for_docking ligand_areas = docking_sidechain/>    <side_chain_for_final ligand_areas = final_sidechain/>    <backbone ligand_areas = final_backbone extension_window = 3/></INTERFACE_BUILDERS><MOVEMAP_BUILDERS>    <docking sc_interface = side_chain_for_docking minimize_water = true/>    <final sc_interface = side_chain_for_final bb_interface = backbone minimize_water = true/></MOVEMAP_BUILDERS><MOVERS>    <Transform name = "transform" chain = "X" box_size = "8.0" move_distance = "5.0" angle = "360" cycles = "5000" temperature = "5" initial_perturb = "1.0"/>    <HighResDocker name = high_res_docker cycles = 1 repack_every_Nth = 1 scorefxn = ligand_soft_rep movemap_builder = docking/>    <FinalMinimizer name = final scorefxn = hard_rep movemap_builder = final/>    <InterfaceScoreCalculator name = add_scores chains = X scorefxn = hard_rep/></MOVERS><PROTOCOLS>    <Add mover_name = transform/>    <Add mover_name = high_res_docker/>    <Add mover_name = final/>    <Add mover_name = add_scores/></PROTOCOLS></ROSETTASCRIPTS>The following RosettaScripts protocol has been used to for DiB1 redesign:<ROSETTASCRIPTS><SCOREFXNS>    <ligand_soft_rep weights = ligand_soft_rep>        <Reweight scoretype = fa_elec weight = 0.42/>        <Reweight scoretype = hbond_bb_sc weight = 1.3/>        <Reweight scoretype = hbond_sc weight = 1.3/>        <Reweight scoretype = rama weight = 0.2/>    </ligand_soft_rep>    <hard_rep weights = ligandprime>        <Reweight scoretype = fa_intra_rep weight = 0.004/>        <Reweight scoretype = fa_elec weight = 0.42/>        <Reweight scoretype = hbond_bb_sc weight = 1.3/>        <Reweight scoretype = hbond_sc weight = 1.3/>        <Reweight scoretype = rama weight = 0.2/>    </hard_rep></SCOREFXNS><SCORINGGRIDS ligand_chain = "X" width = "16">    <vdw grid_type = "ClassicGrid" weight = "1.0"/></SCORINGGRIDS><TASKOPERATIONS>    <DetectProteinLigandInterface name = design_interface cut1 = 6.0 cut2 = 8.0 cut3 = 10.0 cut4 = 12.0 design = 1/></TASKOPERATIONS><LIGAND_AREAS>    <docking_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false minimize_ligand = 10/>    <final_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false/>    <final_backbone chain = X cutoff = 7.0 add_nbr_radius = false all_atom_mode = true Calpha_restraints = 0.3/></LIGAND_AREAS><INTERFACE_BUILDERS>    <side_chain_for_docking ligand_areas = docking_sidechain/>    <side_chain_for_final ligand_areas = final_sidechain/>    <backbone ligand_areas = final_backbone extension_window = 3/></INTERFACE_BUILDERS><MOVEMAP_BUILDERS>    <docking sc_interface = side_chain_for_docking minimize_water = true/>    <final sc_interface = side_chain_for_final bb_interface = backbone minimize_water = true/></MOVEMAP_BUILDERS><MOVERS>    <Transform name = "transform" chain = "X" box_size = "8.0" move_distance = "5.0" angle = "360" cycles = "5000" temperature = "5" initial_perturb = "1.0"/>    <HighResDocker name = high_res_docker cycles = 1 repack_every_Nth = 1 scorefxn = ligand_soft_rep movemap_builder = docking/>    <FinalMinimizer name = final scorefxn = hard_rep movemap_builder = final/>    <InterfaceScoreCalculator name = add_scores chains = X scorefxn = hard_rep/>    <FavorNativeResidue name = favor_native bonus = 1.0/>    <ddG name = calculateDDG jump = 1 per_residue_ddg = 1 scorefxn = hard_rep/>    <PackRotamersMover name = designinterface scorefxn = hard_rep task_operations = design_interface/></MOVERS><PROTOCOLS>    <Add mover_name = transform/>    <Add mover_name = favor_native/>    <Add mover_name = designinterface/>    <Add mover_name = high_res_docker/>    <Add mover_name = final/>    <Add mover_name = calculateDDG/>    <Add mover_name = add_scores/></PROTOCOLS></ROSETTASCRIPTS>S1 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigC).Each datapoint corresponds to one of 5 000 models generated for each round of the docking. Some outliers with high total and/or interface scores are not shown for better visibility of the majority of the data. Insets show overlays of the ligand positions in the 50 best models of the corresponding round of docking . After the third round, docking converged on a single binding pose solution ((TIF)Click here for additional data file.S3 FigThe protocol starts with a small ligand position perturbation followed by optimization of the protein:ligand interface. Amino acids are allowed to change their identity only if it results in a significant decrease in energy, defined through a “favor native” bonus (1 REU in the used protocol). The sequence design step is followed by reevaluation of the ligand position using high resolution docking protocol. The final model is scored and saved for further analysis.(TIF)Click here for additional data file.S4 FigRepresented are data for 35–40 cells from the same field of view across the whole ligand concentration range. Box whiskers indicate standard deviation, horizontal lines within boxes indicate median values, circles indicate outliers.(TIF)Click here for additional data file.S5 Fig1-109 or (B) DiB-RM-split110-177-TagBFP constructs. The signal from green and blue channels, correspondingly. Scale bars are 25 μm.Widefield fluorescence images of HEK293 cells transiently transfected with (A) NeonGreen-DiB-RM-splitN(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigA) using the same DiB1 model that was generated for the initial docking but with deleted E/F loop (-6 amino acids) or (B) using the DiB1 crystal structure-based DiB-RM model starting from the initial (“old”) starting position of the ligand. The M739 chromophore from the co-crystal structure is shown as yellow sticks. All docked chromophores are shown as green lines.Rerun of the chromophore M739 docking ((TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.S2 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.S3 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.


Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants’ occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants’ tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants’ emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant’s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant’s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world. Early on 2020 major viral diagnostic efforts were deployed, including the development of news strategies aiming at (1) reducing the diagnostics time, (2) decrease the costs per assay, (3) providing population-scale solutions, but also (4) presenting a high sensitivity and specificity, notably to discriminate asymptomatic cases. While costs and required time per assay were early on addressed by the development of immune-based tests5. These efforts rely on (1) the incorporation of DNA molecular barcodes during sample preparation; (2) pooling barcoded samples for an “in-one-run” diagnostics assay by the use of massive parallel DNA sequencing; (3) stratification of diagnostics’ outcome with the help of bioinformatics processing. While such strategies demonstrated to up-scale the diagnostics power beyond several thousand candidates per assay, they require to count with a DNA sequencing platform where large instruments (Illumina Sequencers) are driven by specialized technical engineers. As consequence, this strategy remains applicable to highly developed countries (due to the prohibitive costs of the required DNA sequencers), which in addition requires a dedicated pipeline strategy for transporting collected samples towards sequencing centers.With the aim of taking advantage of the viral nucleic acid targeting diagnostics but at the same time increasing the number of candidates’ through-put, strategies based on the use of massive parallel DNA sequencing for population-scale diagnostics were proposed6.Beyond the diagnostics requirements, the identification of the highly transmissible SARS-CoV-2 variant B.1.1.7—originated in the south of England—has strongly accelerated the worldwide interest in tracking SARS-CoV-2 variants’ occurrence. More recently, two other extremely infectious variants, namely the P.1 issued on the Brazilian city of Manaus and the B.1.351 initially detected In South Africa, were described, and unsurprisingly further others are expected to be discovered, notably due to the long period of time on which the pandemic situation is lasting7). Indeed, this technology is widely used for SARS-CoV-2 whole-genome sequencing, notably by the use of the amplicon sequencing protocol developed by the ARTIC Network (https://artic.network/ncov-2019)8. Furthermore, a Covid-19 diagnostics strategy combining Loop-mediated isothermal Amplification (LAMP) with Nanopore sequencing has been also described9.Herein, we describe a proof of concept study for population-scale diagnostics and variants’ tracking using massive parallel DNA sequencing performed with the MinION Oxford Nanopore Technology (ONT). ONT MinION sequencers are well-known for their portability, their reduced cost, and their simplicity of use; thus, representing a suitable strategy for its implementation even in remote places over the world  providing the outcome of the diagnostic in a real-time mode during sequencing; allowing to earn time for diagnostics report, essential in the current context of the pandemic situation. Finally, we have also demonstrated that our strategy is able to perform SARS-CoV-2 variants’ detection across multiple candidates, notably by targeting the SPIKE gene instead of covering the whole viral genome Fig. . This ta10.In summary, the proposed strategy satisfies the current need for counting with a population diagnostic assay, and discriminates between the various SARS-CoV-2 variants, which are recognized to potentially present variable infection properties as well as clinical outcomesWith the aim of performing SARS-CoV-2 diagnostics over multiple candidates with the MinION sequencer, we combined a regular reverse-transcription assay, with a particular PCR amplification allowing the generation of long DNA concatemers. This strategy allows to target short amplicons, like those used on quantitative real-time PCR strategies, but at the same time take advantage of the long fragments sequencing capabilities of the Oxford Nanopore technology.Reverse transcription (RT) is performed with a Covid-19 specific primer, presenting in addition, a unique molecular barcode (20nt length) and a common sequence adapter   and a reverse-oriented sequence for the common Gibson sequence adapter (30nt length) is used together with a Gibson primer . Hence, we have developed a computational pipeline dedicated to (1) collect available fastq files; (2) retrieve Gibson adapter sequences within sequenced molecules; 3) split them into monomers; (4) evaluate the presence of cDNA & PCR molecular barcodes in their flanking regions; (5) align the inner monomer sequences towards the SARS-CoV-2 reference genome; and (6) display the aligned read counts per cDNA/PCR barcode combinations. This pipeline is setup to be reinitiated in an automated time interval (e.g. every 10 min) such that newly available sequences can be cumulated to the previous analyses, thus providing a real-time view of aligned read counts associated with cDNA/PCR barcode combinations  were used for mimicking variable viral RNA copies per assay. Specifically, subsequent dilutions of the synthetic Wuhan-Hu-1 (GeneBankID: MN908947.3) SARS-CoV-2 RNA control sample has been used for reverse transcription, followed by a quantitative PCR assay targeting a region on the E gene of SARS-Cov-2, defined by primer sequences described previously (E-Sarbeco)For Nanopore DNA sequencing-based diagnostic, as described on Fig. Subsequent dilutions of the synthetic SARS-CoV-2 RNA control sample were used for reverse transcription, performed with defined barcoded E-Sarbeco primers , in addition to a negative control sample devoid of viral RNA material (BC37)  we can afford to combine diagnostic and variants’ tracking into a single assay, which per se represents a major progress relative to the current strategies.In contrast to the aforementioned diagnostics effort targeting a short amplicon (E-Sarbeco), variants’ tracking over the whole SPIKE gene uses a random-hexamer sequence for the reverse transcription step, and four primers targeting the SPIKE gene at different regions (~ 1 kb distance among each primer) Fig. A. Like i12 , human RNA—issued from cell lines in culture—has been combined with the synthetic viral RNA samples, such that the random-hexamer primers used for the reverse transcription assay could also provide means to count with a positive control for sample collection. Furthermore, for addressing variants tracking performance, in addition to the Wuhan-Hu-1 SARS-CoV-2 RNA control sample, we have used synthetic viral RNA samples corresponding to the highly transmissible SARS-CoV-2 variant B.1.1.7, first described in the UK in December 2020, as well as the strain HF2393 described as a particular clade circulating in France since January 202012 Fig. C.To perform diagnostics and variants’ tracking assay over multiple pseudo-candidates, we have used eight barcoded random-hexamer primers for reverse transcription BC37-BC44) in presence of different viral RNA titers (consecutive dilutions) and issued from the aforementioned synthetic viral RNA strains  revealed a single common mutation, D614G, previously described within the French strain HF23936. As consequence, counting with methodologies for Covid-19 diagnostics but also variants’ tracking on a population scale remains essential.Despite the current vaccination efforts, the pandemic situation anticipates the propagation of the recently described variants, as well as the emergence of new others, notably due to the major discrepancies between countries to access to optimal vaccination programsCurrently, diagnostics and variants’ tracking are performed in two steps, which per se delays the time for obtaining a complete diagnostic. Furthermore, variants’ tracking relies on full genome sequencing of SARS-CoV-2, which represent an expensive and labor-intensive effort, incompatible with population-scale assays. Finally, most of the variants’ tracking efforts, relying on the use of large and expensive sequencing instruments, requires to centralize samples processing to dedicated institutions, which are rather absent on poor countries.To address all these pitfalls, we propose herein a methodology able to (1) perform SARS-CoV-2 diagnostic and variants’ tracking at the same time; (2) shorten the time for accessing to the outcome of the diagnostic/variants’ detection by the use of a real-time tracking system; (3) take advantage of the compact and affordable Oxford Nanopore MinION DNA sequencing instrument, notably for deploying the aforementioned diagnostics and variants’ tracking platform anywhere over the world.Within this study we have validated the proposed Nanopore DNA sequencing-based diagnostic, by targeting a region on the E gene of SARS-Cov-2 (E-Sarbeco) on a total of 64 pseudo-candidate samples Fig. . Importa13. Illumina instruments being a short fragments sequencing approach, HiSpike requires 42 primer pairs generating amplicons of 400 nucleotides of length for covering the SPIKE region. In contrary, our proposed strategy, based on the use of long DNA fragments, compatible with the Nanopore DNA sequencing technology, relies on 4 primers targeting the SPIKE region, one random primer for the cDNA step and a common Gibson sequence. In fact, a strategy for SARS-CoV-2 diagnostic and variants’ surveillance, dedicated to be deployed anywhere on the world does not only requires to count with low cost instruments, but also involve a reduced number of reagents for allowing its use on a long-standing manner.The proposed variants’ tracking focus on the analysis of the SPIKE gene instead of the whole SARS-CoV-2 genome. In fact, the SPIKE gene has shown to concentrate the majority of the currently discovered variants, which can be rationalized by the fact that it codes for the protein required for the interaction with the human cells. Noteworthy, most of the current vaccines against the Covid-19 are also based on the SPIKE region. Hence, concentrating variants’ tracking towards this gene appears an optimal compromise to increase the number of candidates to screen at once. This philosophy is shared by a recent work describing the HiSpike method, performing variants’ detection within the SPIKE gene with the help of the small Illumina MiSeq instrument14. In all cases, new mutations on the gene SPIKE were observed, further supporting our strategy for targeting this genomic region for variants surveillance.During the time of the final revision of this article, new variants derived from the lineage of B.1.1.7 has gained major attention notably due to the fact that they are vastly spreading in the UK, USA, and other locations For all these reasons, we are convinced that our proposed strategy, accompanied by the dedicated computational solution RETIVAD, can represent a game-changer approach for tracing variants’ progression in the following months.Wuhan-Hu-1 (GeneBankID: MN908947.3); Twist Biosc. ID: Ctrl2.France/HF2393/2020 (EPI_ISL_418227); Twist Biosc. ID: Ctrl7.England/205,041,766/2020 (EPI_ISL_710528); Twist Biosc. ID: Ctrl14 (B.1.1.7).The following Synthetic SARS-CoV-2 RNA control samples, delivered at a concentration of 1 million copies per microliter has been used in this study:11, has been used:Primers used for reverse transcription are composed by (1) a fragment of the Gibson sequenced (redGibs: GAAGAACCTGTAGATAACTCGCTGT), (2) a barcode sequence issued from the ONT PCR Barcoding Expansion 1–96 kit (EXP-PBC096) and (3) the target sequence. For targeting the E-Sarbeco region within the SARS-CoV-2 genome, the reverse primer sequence provided by Corman et al.E_Sarbeco_R: ATATTGCAGCAGTACGCACACAFor SPIKE-centered variants tracing, the target sequence has been replaced by a random hexamer, notably to decrease the number of required primers during the assay.The following barcoded primers targeting the E-Sarbeco region were used for reverse transcription:redGibsBC33_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCAGACTTGGTACGGTTGGGTAACTATATTGCAGCAGTACGCACACAredGibsBC34_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTGGACGAAGAACTCAAGTCAAAGGCATATTGCAGCAGTACGCACACAredGibsBC35_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCTACTTACGAAGCTGAGGGACTGCATATTGCAGCAGTACGCACACAredGibsBC36_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTATGTCCCAGTTAGAGGAGGAAACAATATTGCAGCAGTACGCACACAredGibsBC37_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTGCTTGCGATTGATGCTTAGTATCAATATTGCAGCAGTACGCACACAredGibsBC38_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTACCACAGGAGGACGATACAGAGAAATATTGCAGCAGTACGCACACAredGibsBC39_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCCACAGTGTCAACTAGAGCCTCTCATATTGCAGCAGTACGCACACAredGibsBC40_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTTAGTTTGGATGACCAAGGATAGCCATATTGCAGCAGTACGCACACAThe following barcoded random-hexamer primers were used for reverse transcription:redGibsBC37_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTGCTTGCGATTGATGCTTAGTATCANNNNNNredGibsBC38_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTACCACAGGAGGACGATACAGAGAANNNNNNredGibsBC39_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCCACAGTGTCAACTAGAGCCTCTCNNNNNNredGibsBC40_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTTAGTTTGGATGACCAAGGATAGCCNNNNNNredGibsBC41_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTGGAGTTCGTCCAGAGAAGTACACGNNNNNNredGibsBC42_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCTACGTGTAAGGCATACCTGCCAGNNNNNNredGibsBC43_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCTTTCGTTGTTGACTCGACGGTAGNNNNNNredGibsBC44_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTAGTAGAAAGGGTTCCTTCCCACTCNNNNNN11, has been used:For PCR amplification a second set of barcoded primers were designed presenting the following structure: (1) A reverse-complementary Gibson fragment (revGibs: ACAGCGAGTTATCTACAGGTTCTTCAATGT), (2) a barcode sequence issued from the ONT PCR Barcoding Expansion 1–96 kit (EXP-PBC096), and (3) the target sequence. For targeting the E-Sarbeco region within the SARS-CoV-2 genome, the forward primer sequence provided by Corman et al.E_Sarbeco_F: ACAGGTACGTTAATAGTTAATAGCGTThe following barcoded primers targeting the E-Sarbeco region were used in this study:revGibs-_BC01_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTAAGAAAGTTGTCGGTGTCTTTGTGACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC02_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTCGATTCCGTTTGTAGTCGTCTGTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC03_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTGAGTCTTGTGTCCCAGTTACCAGGACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC04_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCGGATTCTATCGTGTTTCCCTAACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC05_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTCTTGTCCAGGGTTTGTGTAACCTTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC06_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCTCGCAAAGGCAGAAAGTAGTCACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC07_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTGTGTTACCGTGGGAATGAATCCTTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC08_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCAGGGAACAAACCAAGTTACGTACAGGTACGTTAATAGTTAATAGCGTFor SPIKE-centered variants tracing, the reverse-complementary Gibson sequence has been excluded, notably because the PCR amplification performed over random-hexamer generated cDNA templates generate large fragments (> 1 kb). In contrary, the amplicon product issued from E-Sarbeco targeting oligonucleotides gives rise to short amplicons—compatible with quantitative PCR assays, thus requiring concatemerization prior nanopore sequencing.With the aim of covering the SPIKE gene (~ 3.8 kb), the following four primers targeting the SPIKE region with ~ 1 kb interval within them were designed:Spike1_F: AGGGGTACTGCTGTTATGTCTSpike2_F: TGCACTTGACCCTCTCTCAGSpike3_F: GCAGGCTGTTTAATAGGGGCSpike4_F: TGCAGACATATGTGACTCAACAThe following Spike targeting primers including the corresponding barcode sequences were used for this study:BC01_Spike_1_F: AAGAAAGTTGTCGGTGTCTTTGTGAGGGGTACTGCTGTTATGTCTBC02_Spike_1_F: TCGATTCCGTTTGTAGTCGTCTGTAGGGGTACTGCTGTTATGTCTBC03_Spike_1_F: GAGTCTTGTGTCCCAGTTACCAGGAGGGGTACTGCTGTTATGTCTBC04_Spike_1_F: TTCGGATTCTATCGTGTTTCCCTAAGGGGTACTGCTGTTATGTCTBC01_Spike_2_F: AAGAAAGTTGTCGGTGTCTTTGTGTGCACTTGACCCTCTCTCAGBC02_Spike_2_F: TCGATTCCGTTTGTAGTCGTCTGTTGCACTTGACCCTCTCTCAGBC03_Spike_2_F: GAGTCTTGTGTCCCAGTTACCAGGTGCACTTGACCCTCTCTCAGBC04_Spike_2_F: TTCGGATTCTATCGTGTTTCCCTATGCACTTGACCCTCTCTCAGBC01_Spike_3_F: AAGAAAGTTGTCGGTGTCTTTGTGGCAGGCTGTTTAATAGGGGCBC02_Spike_3_F: TCGATTCCGTTTGTAGTCGTCTGTGCAGGCTGTTTAATAGGGGCBC03_Spike_3_F: GAGTCTTGTGTCCCAGTTACCAGGGCAGGCTGTTTAATAGGGGCBC04_Spike_3_F: TTCGGATTCTATCGTGTTTCCCTAGCAGGCTGTTTAATAGGGGCBC01_Spike_4_F: AAGAAAGTTGTCGGTGTCTTTGTGTGCAGACATATGTGACTCAACABC02_Spike_4_F: TCGATTCCGTTTGTAGTCGTCTGTTGCAGACATATGTGACTCAACABC03_Spike_4_F: GAGTCTTGTGTCCCAGTTACCAGGTGCAGACATATGTGACTCAACABC04_Spike_4_F: TTCGGATTCTATCGTGTTTCCCTATGCAGACATATGTGACTCAACAReverse transcription assays were performed using SuperScript™ IV Reverse Transcriptase ) with a standard reaction volume of 20ul including 0.5ul of super RNAseIn , 1ul DTT (0.1 M), 4 ul SSIV buffer (5X), 0.5 ul dNTP (10 mM each), 1 ul of synthetic SARS-CoV-2 RNA, 1 ul of 2uM gene-specific-barcoded oligonucleotide  or 1 ul of 50uM random hexamer-barcoded oligonucleotide . For assays including random hexamer-barcoded primers, 1ul of human RNA  were added to the assay together to the synthetic SARS-CoV-2 RNA and prior adding the random hexamers.Synthetic SARS-CoV-2 RNA was added at a copy number of 1 million , 100 thousand (d.f: 0.1), 10 thousand (d.f: 0.01) and 1 thousand (d.f: 0.001) per reaction respectively. Note that these consecutive dilutions give rise after downstream dilutions to the number of copies of 10 thousand, 1 thousand, 100 and 10 respectively, analyzed by quantitative PCR Fig. B.Multiple reverse transcription assays—each of them performed in presence of a defined barcoded primer—were incubated at 50 °C during 30 min, then at 80 °C 10 min. Barcoded cDNA samples were pooled (8 samples at once) and cleaned with SPRIselect reagent  with a ratio of 0.5x. Cleaned cDNA is recovered on 80ul . 40ul of this pooled & cleaned cDNA will be used for PCR amplification , while the remaining material is used for quantitative PCR evaluation.Barcoded-cDNA material has been validated by quantitative PCR assay with the following primer sequences:Primers targeting region on E gene of SARS-Cov-2 :Human_RNASE_P-F: AGATTTGGACCTGCGAGCGHuman_RNASE_P-R: GAGCGGCTGTCTCCACAAGTQuantitative PCR assays were performed with QuantiTect SYBR Green PCR Kit  over a 1/10 diluted cDNA material.PCR amplification targeting the E-Sarbeco amplicon is performed on 25ul final volume including 12.5ul of Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher ref: F565), 2.5ul of 1uM barcoded-E-Sarbeco targeting primer (BC1-8), 2.5ul of 1uM short Gibson primer (AGAACCTGTAGATAACTCGCTGT) and 5ul of the pooled & cleaned cDNA.98 °C 30 s98 °C 10 s61 °C 20 s72 °C 30 sRepeat 2–4 steps 25×98 °C 10 s61 °C 20 s72 °C 2 minRepeat 6–8 steps 20×72 °C 10 minKeep at 12 °CFor short-amplicon E-Sarbeco PCR amplification the following thermal cycling program is used:For PCR amplification targeting SPIKE and E-Sarbeco on random-hexamers generated cDNA material the assay is performed with a pool of barcoded SPIKE primers and shortGibson. For 100ul mix 10ul of barcoded primer Spike1 (10uM), 10ul of Spike2 (10uM), 10ul of Spike (10uM), 10ul of Spike4 (10uM) and 40ul of ShortGibson (10uM) completed with distilled water is prepared. Then the PCR amplification is performed on 25ul final volume including 12.5ul of Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher ref: F565), 1.25ul of pooled barcoded-SPIKE & shortGibson primers  and 5ul of pooled & cleaned cDNA.98 °C 30 s98 °C 10 s61 °C 45 s72 °C 2 minRepeat 2–4 steps 39×72 °C 10 minKeep at 12 °CThe following thermal cycling program is used:In both cases (E-Sarbeco Concat-PCR or SPIKE PCR amplification), PCR products were verified by TapeStation automated electrophoresis .Multiple PCR assays are performed in presence of different barcoded primers, such that the combination with the barcoded cDNA material allows to generate a large complexity. Specifically, for E-Sarbeco targeting assay, 8 barcoded contact-PCR assays are pooled (8 × 25 = 200ul) and cleaned with SPRIselect reagent  with a ratio of 0.5×. Similarly, for SPIKE and E-Sarbeco PCR assays issued from random hexamer cDNA material, 4 barcoded PCR assays for each target regions  were pooled (4 × 2 × 25 = 200ul) and cleaned with SPRIselect reagent  with a ratio of 0.5x.Pooled & cleaned material is recovered on 50ul and used for Nanopore sequencing library preparation (Nanopore Ligation Sequencing Kit: SQK-LSK109). DNA libraries were sequenced on Nanopore FLO-MIN106D flowcells (R9) with the MinION Mk1C instrument .https://pypi.org/project/regex) with maximum 4 mismatches accepted by default); (2) split sequenced reads on monomers—delimited by the presence of the identified Gibson sequences; (3) search for barcode sequences introduced during the reverse transcription ; (4) search for barcodes introduced during the PCR amplification ; (5) collect the sequences between the cDNA and PCR-associated barcodes for their alignment towards the SARS-CoV-2 reference genome . Considering that steps (3–5) are performed within the retrieved monomers in step (2), RETIVAD matches cDNA/PCR barcode combinations to aligned outcomes, thus providing a diagnostic outcome for the associated pseudo-candidate. Due to the low number of sequences retrieved at intervals of 10 min, and the relative short size of the SARS-CoV-2 genome (~ 29 kb), RETIVAD is able to provide such diagnostic outcome within such 10 min interval. Hence, at the next round of data collection, RETIVAD reiterates on the aforementioned steps over the newly collected fastq files and appends the outcome to the previous results. The continuous gain on aligned read-counts per region of interest  is visualized within scatterplot view (png file) updated during the processing  software. RETIVAD collects each ten minutes the fastq files generated during sequencing from the Mk1C instrument and (1) search for Gibson sequences within the sequenced reads (regex query (racon package (version 1.4.20) is used for raw de novo DNA assembly, followed by the alignment with Minimap2 (version 2.17). Medaka (version 1.2.5) is used to create a consensus sequence and variant calls from the aligned data . RETIVAD generates BAM files per cDNA/PCR barcode combination harboring base conversion information and their associated coverage . Furthermore, summary files per cDNA/PCR barcode combination are also generated, providing the genomic position in which the base conversion has been detected, the identity of the converted bases as well as their associated coverage. Like in the case of the diagnostics processing, variants detection is performed in real time, thus providing the possibility to the user to visualize the results and decide whether sequencing requires still to be performed or whether the collected information is sufficient to conclude the diagnostics/variants tracing for the corresponding candidates.In addition to the diagnostic outcome, RETIVAD has been designed for performing real-time variants detection. For it, the RETIVAD generates a final summary report for the diagnostics outcome when no new fastq files are available after 40 min from the last data collection and processing.Supplementary Information.


HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease.Heterozygous mutations in  Summary: A novel established mouse model carrying a heterozygous splicing human mutation in the Hnf1b gene exhibits phenotypes similar to those of patients with renal cysts and diabetes disease. HNF1B gene are the cause of a complex human syndrome known as renal cysts and diabetes , characterized by early onset of diabetes and developmental abnormalities of the kidney, genital tract and pancreas, as well as a variety of renal, liver, pancreas and biliary dysfunctions . Intragenic mutations map predominantly in the DNA binding domain (exon-2 and exon-4), while whole-gene deletions (1.3 Mb deletion at chromosome 17q12) account for up to 50% of the HNF1B variants. HNF1B mutant carriers exhibit a highly variable phenotype, both between and within families. No clear genotype-phenotype correlations were observed for the type or location of mutations, and haploinsufficiency has been the main underlying disease proposed mechanism.More than 150 eletions . These mHNF1B mutations is severe non-diabetic renal disease. HNF1B mutations are also the most common monogenic causes of developmental kidney disease, being part of a spectrum of malformations known as congenital anomalies of the kidney and urinary tract  branching and the induction of nephrogenesis  . Moreoveutations , this moSp2/+Hnf1b] exhibited bilateral cysts and tubular dilatations as well as glomerular cysts from embryonic day (E)15, along with rare cases of genital tract abnormalities and extrarenal manifestations similar to those described in human HNF1B mutant carriers. Unlike previous mouse mutants heterozygous for a null allele, in which the HNF1B protein levels remained either unchanged or even increased  ncreased , the HNFHnf1b ablation were relatively insensitive. Postnatal analyses revealed several renal abnormalities, ranging from few clusters of glomerular cysts and microcysts to hydronephrosis and, rarely, multicystic dysplasia. Further urine proteomic analyses uncovered a particular signature of differentially excreted peptides in Hnf1bSp2/+ mice, exhibiting several similarities to the urinary peptide signature reported in pediatric RCAD patients  was sensitive to reduced HNF1B levels at different embryonic stages, whereas the previously identified targets upon patients .Hnf1b dosage in this mouse model differentially affects the expression of target genes, leading to the onset of disease phenotypes.Our results highlight that reduced Fig. S1A), thus reproducing the c.544+1G>T (<IVS2nt+1G>T) human mutation  using RNA from Hnf1bSp2/+ heterozygotes and wild-type (WT) kidneys and primers located in exon-1 and exon-3. Sequence of the PCR products in Hnf1bSp2/+ mutants indicated the production of the two expected Hnf1b spliced isoforms A and B and four additional novel transcripts, present at low levels, corresponding to the isoforms A and B in which were deleted either exon-2 or the last 32 bp of exon-2 through the activation of a near cryptic splice donor site . The same pattern of alternative splicing in Hnf1bSp2/+ heterozygous mice was observed at different stages .To define the consequences of this mutation on Fig. S1C), variants lacking the last 32 bp of exon-2 have not been described  using primers located in the ATG translational site and the last 32 bp of exon-2, thus allowing the detection of transcripts produced only by the normal Hnf1b allele (Table S2). We found that, in Sp2/+Hnf1b, the Hnf1b transcripts were significantly decreased by 30-42% relative to WT levels, in particular during embryonic stages. In adults, the reduction in transcript levels was highly variable and modest without reaching significance . Thus, these truncated proteins are either very unstable or not produced as previously described in several dominant mutations associated with premature termination codons (PTCs) (see Discussion). Unlike previously characterized heterozygous mutants, we found a significant decrease in HNF1B protein levels in the Sp2/+Hnf1b mutants, not only at embryonic stages but also in adults , although in some cases mutant embryo kidneys exhibited dilated Bowman's capsules . Subsequently, from E15.5, we reproducibly observed bilateral cysts, including glomerular and tubular cysts in the cortico-medullar regions . Up to E14.5, heterozygous embryos developed apparently normally . A summary of histological renal phenotypes observed from E18.5 to postnatal day (P)0 is shown in Table S1A.We then examined the influence of mouse strain susceptibility on the disease phenotype by backcrossing the om E15.5 G-N and a kidneys ; Fig. S4Table S1B). Heterozygous mutants in both backgrounds were able to reproduce, but they were less fertile than WT littermates. Further postnatal analysis revealed rare cases of genital tract abnormalities  in either C57BL/6N or 129/sv backgrounds. Heterozygous mutants also exhibited several pancreatic dysfunctions, including glucose intolerance and pancreatitis, a phenotype that will be described elsewhere  or attenuate (129/sv) the phenotype, further contributing to variability in phenotypic manifestations.These data show that Hnf1b is known to be required for early UB branching and initiation of nephrogenesis . Notably, the expression of Pax2, the transcription of which in the collecting system depends on Hnf1b , or at later stages . Nascent nephrons appeared normally induced as indicated by the presence of normally shaped comma- and S-shaped bodies  and WT1-stained glomerular podocytes . Mutant kidneys without overt hydronephrosis were examined to visualize the collecting duct network and medullar nephron tubules. The expression of HNF4A, a marker of early PTs and a target of HNF1B , suggesting defective differentiation of PTs. Notably, HNF4A staining, which correlated with the reduced levels of HNF1B , in contrast to HNF4A expression, which showed only a partial restoration at E17.5  or P0 . Consistent with these observations, the mature PT marker LTA was not expressed up to E16.5 . However, Sp2/+Hnf1b PT clusters that were LTA+ both at E17.5 and P0 exhibited an unequal distribution and remained reduced in size . Interestingly, the Na-K-Cl co-transporter , expressed in the thick ascending limb (TAL) of the loop of Henle, stained most of Sp2/+Hnf1b medullar cysts and tubular dilatations at E17.5 and P0 . Distal tubules labeled by SLC12A3 (NCC) showed only rare and mild dilatations (data not shown).The expression of HNF1A, restricted to mature PTs, was similarly reduced at E16.5 B′. Its eto E16.5 F′ and beSp2/+Hnf1bcollecting ducts, stained either by AQP2  or pancytokeratin (CK) , were mainly devoid of dilatations. Intriguingly, they were not stained by Dolichos biflorus lectin (DBA), either during development or in postnatal life , uncovering impaired polarization of glycoconjugates in mutant collecting duct cells. This observation evoked an altered differentiation status of collecting duct cells, because previous studies have shown that UB cells begin to express DBA-binding glycoconjugates once they differentiate into stalks   .Sp2/+Hnf1b non-cystic glomeruli displayed the normal layer of podocytes .Without evidence of urinary tract obstruction, we further examined HNF1B, HNF4A and WT1 immunostainings on serial embryonic sections, focusing on glomeruli with different degrees of Bowman's capsule expansion. We reproducibly observed that cystic glomeruli were surrounded by a decreased number of PTs that were often dilated or cystic H. Immunoodocytes F,I. In mHNF1B human mutant fetuses showing a very severe cystic renal phenotype  exhibited a global decrease and partial disorganization in E15.5 heterozygous kidneys, both in dilated and non-dilated tubules K,M. In mFig. S6A). However, at later stages, the number of proliferating cells in Sp2/+Hnf1b normal tubular structures exhibited a 1.84-fold increase relative to WT tubules. Consistent with the enhanced proliferation associated with cystic expansion in autosomal dominant polycystic kidney disease . No changes were observed in the number of apoptotic cells assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (not shown).Further analysis of proliferation, using the mitosis marker phosphorylated histone H3, showed comparable numbers of proliferating cells in E14.5 renal tubules of heterozygous mutants and WT  on WT and heterozygous mutant kidneys at E14.5  and at disease stages  as detailed in the Materials and Methods.P-value <0.05. Remarkably, several genes were differentially expressed with log2FC>−1 at all stages (Table S3), while only a few differentially expressed upregulated genes were detected. We found an increased number of downregulated genes from E14.5 to E17.5 followed by a partial decrease at P1, in agreement with a partial restoration of the expression of several PT markers. Accordingly, the numbers of downregulated genes shared between stages increased to reach 107 genes between E17.5 and P1 .Because heterozygous mutants still expressed HNF1B, we considered an absolute fold-change cutoff value of > or <0.5 log2FC, with an adjusted Table S3, File 6). At E17.5 and P1, downregulated genes included drug-metabolizing enzymes  and more than 50 SLC-transmembrane-transporter genes .Downregulated genes with greater fold changes were predominantly expressed in early and maturing PTs and to a lesser extent in primitive loops of Henle and distal tubules (Table S4).Gene ontology (GO)-term analyses  highlighHnf4a, Tmem27 (Cltrn), Cubn, Spp1, Spp2, Pah, Kcnj1, Pdzk1 and several SLC-transmembrane transporter genes, representing the subset of targets that appeared particularly sensitive to reduced HNF1B protein levels (Table S3). Interestingly, many of the genes downregulated at E17.5/P1 and regulated by HNF1B were also found strongly decreased in Hnf4a mutant P1 kidneys . By contrast, the expression of previously identified targets strongly reduced upon Hnf1b ablation, including Wnt9b, Pax2, Pkd2, Bicc (Bicc1), Tg737 (Ift88), Crb3, Kif12, Cys1, Glis2 and Glis3, were either modestly decreased or not affected. These results were further validated by qRT-PCR . Interestingly, the well-known targets Umod, Tmem27 and Pkhd1 (Table S3).The regulatory sequences of most downregulated genes contained HNF1 consensus binding sites. Consistently, specific HNF1B recruitment to the regulatory sequences of many of them has been reported by chromatin immunoprecipitation (ChIP)-PCR or ChIP sequencing . This ocular phenotype remains undefined. It may be linked to the Rd8 mutation of the Crb1 gene present in the strain C57BL/6N , with the lacZ gene and the SV40 polyadenylation sequence and neomycin resistance cassette, replacing the first exon of Hnf1b (Hnf1btm1Sce) and C57BL/6N (C57BL/6N-Hnf1btm1Sce) backgrounds are available at EMMA  together with the phenotype description. The +/−Hnf1a mice were provided by Frank Gonzalez    and mainAnimal care and the experimental protocols were approved by and conducted in accordance with French and European ethical legal guidelines and the local ethical committee for animal care , respecting the 3R rules.Paraffin-embedded human fetal tissues were obtained from family members with informed consent approved by the Ethics committee of Debré Hospital , as previously reported in Fbp and Spp2 cRNA probes were generated by PCR . Embryos and postnatal kidneys up to 2 months of age were fixed with 60% ethanol/11% formaldehyde and 10% acetic acid. Adult kidneys (>2 months) were fixed in alcoholic Bouin (Duboscq-Brasil) solution and paraffin sections were used for H&E histological analysis and immunohistochemistry. Antibody staining on paraffin sections was performed as described  and washed in ice-cold PBS. WT and heterozygous mutant embryos were from the same litter. Adult kidneys were cut sagittal: one-half was used for histology, one-quarter for RNA extraction and the other quarter for protein extraction. Total RNA was extracted using an miRNeasy Mini Kit (Qiagen). Samples were treated with Dnase1 on the columns according to the manufacturer's instructions, and 250-500 ng was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) and the Step-One Plus system (Applied Biosystems), as described . The priFig. S1. PCR products were resolved in 2% agarose/TBE ethidium bromide gels and photographed using a GELDOC documentation system. Densitometry quantification was performed with ImageJ software. Primer sequences used were Gapdh for normalization and, for Hnf1b, vATG and v695 (Table S2).Total RNA from microdissected kidneys was extracted and subjected to semiquantitative RT-PCR as described  with theHnf1b allele are listed below:The cDNA sequences of spliced isoforms and the encoded truncated proteins from the mutated CGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGCGAGGACCCGTGGAGGGCTGCCAAAATGATCAAGGGATACATGCAACAGCACAATATCCCCCAGAGGGAGGTGGTCGATGTCACAGGCCTGAACCAATCCCACCTCTCTCAACACCTCAACAAGGGCACCCCCATGAAGACCCAGAAGAGAGCTGCCCTGTACACTTGAgATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLSEDPWRAAKMIKGYMQQHNIPQREVVDVTGLNQSHLSQHLNKGTPMKTQKRAALYT*VQPDTAAaAGTTCAACCAGACAGTCCAGAGCTCTGGAAACATGACAGACAAAAGCAGTCAGGATCAGCTGCTGTTTCTCTTTCCAGAGTTCAGTCAACAGAACCAGGGGCCTGGGCAGTCGGAGGACACCTGCTCCGAGCCCACCAACAAGAAGATGCGCCGCAACCGGTTATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACCGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLRVQPDSPELWKHDRQKQSGSAAVSLSRVQSTEPGAWAVGGHLLRAHQQEDAPQPV*TAAaAGTTCAGTCAACAGAACCAGGGGCCTGGGCAGTCGGAGGACACCTGCTCCGAGCCCACCAACAAGAAGATGCGCCGCAACCGGTTATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACCGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLRVQSTEPGAWAVGGHLLRAHQQEDAPQPV*Note that the STOP codons of putative encoded proteins are followed by a purine, which is predicted to positively influence translational termination efficiency .GTACGTCAGAAAGCAACGGGAGATCCTCCGACCGAGGACCCGTGGAGGGCTGCCAAAATGATCAAGGGATACATGCAACAGCACAATATCCCCCAGAGGGAGGTGGTCGATGTCACAGGCCTGAACCAATCCCACCTCTCTCAACACCTCAACAAGGGCACCCCCATGAAGACCCAGAAGAGAGCTGCCCTGTACACTTGUnderlined text shows the 32 bp of exon-2 spliced out of mouse exon-2.Human embryonic kidney (HEK) 293 cells were maintained and transiently transfected as described , with eig. The cellular pellet was resuspended into ice-cold HNB buffer , frozen and thawed four times in liquid N2, and centrifuged for 10 min at 11,180 g. The supernatant containing the whole-cell extracts was frozen in liquid N2 and kept at −80°C.Transfected cells were washed with ice-cold PBS plus Roche protease inhibitors, scraped, transferred into Eppendorf tubes and centrifuged for 5 min at 1006 n=3) different litters were pooled, frozen in liquid N2 and lysed in ice-cold lysis buffer using 23 g and 26 g syringes. Adult kidney pieces were frozen in liquid N2, reduced to a fine powder under liquid N2 and further lysed in lysis buffer using a syringe. Lysis buffer contained 15% glycerol, 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP40, 3 mM sodium pyrophosphate and 50 mM sodium fluoride, with complete protease inhibitor cocktail (Roche) added before using. Samples were centrifuged for 15 min at 18,894 g at 4°C, and the supernatant was frozen in liquid N2 and kept at 80°C. Protein concentration was determined using a Pierce™ BCA Protein Assay kit. Whole-cell extracts containing 20-30 μg of protein were prepared in SDS sample buffer and subjected to SDS-PAGE . After the proteins were transferred onto a 0.2 µm nitrocellulose membrane (Bio-Rad) and blocked with TBST , immunoblotting was performed by overnight incubation in TBST 1% skim milk buffer with a rabbit polyclonal antibody against HNF1B (1:500) previously validated (Table S7). Positive HNF1B bands were detected by chemiluminescence . Western lightning Plus-ECL Perkin Elmer was used to detect α-tubulin. Images were captured with G-BOX Syngene Europe, Chemiluminescence Image Capture software and quantified by Image J. WT and HNF1B proteins normalized by α-tubulin expression were quantified from each litter. A WT sample was assigned a 100% value and the other samples of the same litter WT and heterozygotes were referred to as a percentage of this value. This allowed the comparison of different litters of a given embryonic stage. Statistical significance was determined using unpaired Student's t-test, using Prism 6.00 . P<0.05 was considered significant.The two kidneys of each embryo of at least three . Twenty-four-hour urine samples collected under mineral oil to avoid evaporation were obtained at baseline in individual metabolic cages, after 2-3 days habituation. Each 24 h, animals were weighed and food intake, water intake, urine volume and fecal weight recorded. Blood was sampled by retro-ocular puncture, in general 2 weeks after being in the metabolic cages, and plasma samples were kept at −80°C. The urinary concentration ability was tested after 22 h of water deprivation. Urinary creatinine, urea and electrolytes, plasma urea, creatinine, Mg2+, AST and ALT were measured on an Olympus AU400 Chemistry Analyzer . Osmolality was measured using a vapor pressure osmometer .Urine and plasma were obtained from age- and sex-matched heterozygous and WT mice . They were housed in a light- and temperature-controlled room with t-test or unpaired Student's t-test with Welch correction were used for statistical analysis. P<0.05 was considered significant.Data are presented as mean±s.e.m. Unpaired Student’s Sp2/+Hnf1b and 18 WT mice from the age of 3, 8, 12 and 17 months. Urine from WT and mutant mice  was collected by spontaneous voiding or after animals were placed individually in metabolic cages as described. The total volume of 24-h urine was aliquoted and frozen at −80°C. A 150-µl sample of mouse urine was diluted with the same volume of urea buffer . Then, 150 µl of the urine samples was ultrafiltrated, desalted, lyophilized and resuspended for proteome analysis as described  online coupled to a micrOTOF II MS , as described . For norHnf1bSp2/+ mutants and WT embryos in the C57BL/6N background were microdissected from the same litter. This requirement limited the number of samples used in general to two WTs and two heterozygous mutants, independently of the sex. Note that we found a similar phenotype in males and females. The stages analyzed included E14.5  and at disease stages E15.5, E17.5 and P1. Note that at E15.5 we performed deep sequencing from pooled samples of three WT and three Sp2/+Hnf1b (six kidneys each sample). RNA-seq of E14.5, E15.5 and P1 samples was performed at the Alexander Fleming Institute, Genomics Facility, Greece; RNA-seq of E17.5 samples was performed at Fasteris, Switzerland (Table S8).The two kidneys from heterozygous ® mRNA DIRECT™ Micro Kit (ThermoFisher Scientific). mRNA was digested with RNase III, purified, hybridized and ligated to Ion Adaptors (ThermoFisher Scientific), reverse transcribed, barcoded and amplified, using an Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific). RNA-seq was performed on an Ion Proton™ System (ThermoFisher Scientific), according to the manufacturer's instructions. The prepared libraries were quantified and pooled together in duplicates at the required concentration. The pools were then processed on a OneTouch 2 instrument and enriched on a OneTouch ES station. Templating was performed using an Ion PI™ Template OT2 200 Kit (ThermoFisher Scientific) and sequencing with an Ion PI™Sequencing 200 Kit on Ion Proton PI™ chips (ThermoFisher Scientific) according to commercial protocols. The resulting RNA-seq BAM files were analyzed with the Bioconductor package metaseqR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html).RNA from microdissected kidneys was extracted by Tryzol, using a Qiagen miRNA mini kit for the extraction of total RNA and miRNAs. The quality of the RNA samples was assessed on an Agilent Bioanalyzer system using an RNA 6000 Nano Kit (Agilent Technologies) and RNA with a Ring higher than 8 was used. Then, 1-2 μg of total RNA was used for mRNA isolation using a DynabeadsmetaseqR , applyinin vitro samples to generate a library of short inserts (the DNA Colonies Template Library). The library was sequenced on an Illumina HiSeq 2000. For each lane, 130-150 million DNA colonies producing pass filter sequences were assured. The read lengths are 1×50 bp or 1×100 bp for single-read runs using the forward sequencing primer. The inserts can also be sequenced from both ends using a ‘forward’ and a ‘reverse’ sequencing primer, generating paired reads of 2×100 bp. The data were processed using bioinformatics tools to extract biologically useful information. To homogenize mRNA-seq comparisons, RNA-seq files were all analyzed as described above (High-throughput DNA sequencing using Illumina technology consisted of processing ed above .