Ethics<\/p>\n
Human peripheral blood was obtained from de-identified healthy donors through the NIH Clinical Center, Department of Transfusion Medicine, Research Blood Donor Program, under a protocol approved by the NIH Institutional Review Board (IRB no. 99CC0168). All donors provided written informed consent before participation.<\/p>\n
Cell culture<\/p>\n
HeLa cells (American Type Culture Collection (ATCC), CCL-2) were cultured in DMEM (Thermo Fisher Scientific, 10313021) supplemented with 2\u2009mM L-glutamine (Thermo Fisher Scientific, 25030164) and 10% bovine calf serum (BCS, HyClone, SH30073.03; DMEM-BCS). THP-1 cells (ATCC, TIB-202) were cultured in RPMI 1640 (Thermo Fisher Scientific, 21870092) supplemented with \u03b2-mercaptoethanol (55\u2009mM, Sigma, M3148), 2\u2009mM L-glutamine and 10% FBS (Seradigm, FBS, 97068-085, RPMI-primary). THP-1 cells were differentiated into M0 macrophages through the addition of phorbol 12-myristate 13-acetate (PMA, 162\u2009nM, Sigma-Aldrich, P1585) with 1.5\u2009\u00d7\u2009106 cells per 6-well plate (for protein) or 0.375\u2009\u00d7\u2009106 cells per 12-well plate (for RNA and luminescence) for 16\u201324\u2009h. THP-1-derived M0 cells were cultured in RPMI-primary medium for 24\u2009h before transfection. HeLa and THP-1 cells were not authenticated. MEFs from a frozen cryovial were thawed and cultured in DMEM with 15% FBS and 1% l-glutamine. Primary monocytes were isolated from human peripheral blood using an EasySep Direct Human Monocyte Isolation kit (Stem Cell Technologies, 19669) according to the manufacturer\u2019s instructions, followed by the addition of 25\u2009mM HEPES (Quality Biological, 118-089-721), 50\u2009ng\u2009ml\u20131 recombinant human IL-4 (Peprotech, 200-04) and 50\u2009ng\u2009ml\u20131 recombinant human GM-CSF (Sigma-Aldrich, GF-304) in RPMI primary medium to generate MoDCs.<\/p>\n
The SINAP technology relies on several auxiliary proteins: scFv\u2013sfGFP to label the nascent peptides, MCP\u2013RFP to label the RNA and the E3 ligase TIR1 from Oryza sativa (OsTIR1) for the auxin-inducible degron to deplete the mature proteins29<\/a>. Two U-2 OS cell lines expressing these auxiliary proteins were used in this study: one for live-cell imaging (scFv\u2013sfGFP, OsTIR1 (ref. 51<\/a>) and MCP\u2013RFP\u2013CAAX), and the other for fixed-cell (scFv\u2013sfGFP, OsTIR2 (ref. 52<\/a>)) experiments. These U-2 OS cells (ATCC, HTB-96) were maintained in DMEM\u2013FBS (Corning, 10-013-CM and Millipore Sigma, F4135), 100\u2009U\u2009ml\u20131 penicillin and 100\u2009\u00b5g\u2009ml\u20131 streptomycin (Millipore, P0781). Cells were cultured at 37\u2009\u00b0C in a 5% CO2 incubator and passaged approximately every 3\u2009days. Monthly mycoplasma contamination testing was performed to ensure sterility.<\/p>\n Flow cytometry<\/p>\n The following antibodies were used for flow cytometry experiments: PE-conjugated anti-Hu\/NHP CD25 antibody ((CD25-4E3), eBioscience, 12-0257-42), APC-conjugated anti-HuCD14 antibody ((61D3), eBioscience, 17-0149-42), PerCP\/cyanine5.5-conjugated anti-human CD11c antibody ((3.9), BioLegend, 301624) and PE-conjugated anti-CD209 (DC-SIGN) antibody ((9E9A8), BioLegend, 330105). In brief, 0.1\u20131\u2009\u00d7\u2009106 cells were stained in 100\u2009\u00b5l staining buffer (1% FBS in PBS) for 30\u2009min at room temperature in the dark, washed twice with PBS and resuspended in 1% paraformaldehyde containing staining buffer. Flow cytometry acquisition was performed on a BD FACSymphony A5 and data were examined using FACSDiva software (v.9.3.1, BD Bioscience). Data were further analysed using FlowJo (v.10.8.1, BD).<\/p>\n Cloning<\/p>\n To generate a 5\u00d7C-stretch in the N-terminally Flag-tagged FLuc sequence3<\/a>, a PCR strategy was used to introduce synonymous mutations of proline 197 and 198 (Supplementary Table 1<\/a>). After confirmation of the mutation by Sanger sequencing, WT-5\u00d7C FLuc was back cloned into the original (WT-5\u00d7U FLuc) plasmid with BsrGI (NEB, R3575) and Bsu36I (NEB, R0524). Single-nucleotide insertion next to the C-rich sequence was accomplished by inserting a mutated DNA fragment (IDT) using BsrGI and Bsu36I. The sequence for the C=U NanoLuc reporter was purchased as a DNA fragment (IDT) and cloned using BbsI-HF (NEB, R3539) and BsmI (NEB, R0134).<\/p>\n In vitro transcription and polyadenylation<\/p>\n Linearized and purified plasmid DNA (purchased from Genscript; see Supplementary Table 1<\/a> for sequences) was IVT with T7 polymerase (NEB, M0251L) or G47A+884G mutant T7 (gift from Y.-X. Wang\u2019s Laboratory) in combination with inorganic pyrophosphatase (Escherichia\u2009coli, NEB, M0361L). Co-transcriptional capping was achieved with CleanCap AG (TriLink, N-7113) according to the manufacturer\u2019s instructions (TriLink). SINAPs reporter mRNAs were post-transcriptionally polyadenylated with E.\u2009coli poly(A)polymerase (NEB, M0276) supplemented with murine RNase inhibitor (NEB, M0314) for 30\u2009min at 37\u2009\u00b0C according to the manufacturer\u2019s instructions. For modified transcripts, ac4CTP (Jena Bioscience, NU-988L) or m5CTP (TriLink, N-1014-5) replaced CTP, and m1\u03a8TP (TriLink, N-1081) or \u03a8TP (TriLink, N-1019-5) replaced UTP in the reaction mix. Purification of polyadenylated RNA was achieved by magnetic separation using RNAClean XP beads (Beckman Coulter, A63987) according to the manufacturer\u2019s instructions. Denaturing agarose gel electrophoresis was performed using NorthernMax 10\u00d7 Denaturing gel buffer (Thermo Fisher Scientific, AM8676). Gels were stained after electrophoresis using SYBR Green\u2009II RNA gel stain (1:10,000 in TBE, Thermo Fisher Scientific, S7564) for 30\u2009min in the dark to visualize RNA. ac4C-modified IVT mRNAs were diluted before gel loading to account for increased staining intensity.<\/p>\n Nucleoside mass spectrometry<\/p>\n Modified and unmodified IVT mRNAs (100\u2009ng each) were digested in 35\u2009\u00b5l using snake venom phosphodiesterase (0.2\u2009U, Abnova, P5263), calf intestinal phosphatase (2\u2009U, Promega, M1821) and benzonase (2\u2009U, Millipore Sigma, E1014) prepared with 1\u2009mM MgCl2 (Quality Biological, 351-033), 5\u2009mM Tris (pH\u20098, Invitrogen, AM9855G), 10\u2009nmol butylated hydroxytoluene (Sigma-Aldrich, B1378) and 5\u2009\u00b5g tetrahydrouridine (Calbiochem, 584222) (modified from a previously described method53<\/a>) and incubated for 2\u2009h at 37\u2009\u00b0C. Samples were diluted with 15\u2009\u00b5l LC\u2013MS buffer\u2009A (0.0075% formic acid (Supelco, 5330020050) in ultrapure water) and filtered for 35\u2009min at 3,273g and 4\u2009\u00b0C through 0.2\u2009\u00b5m Supor AcroPrep Advance 96-well plates (Cytiva, 50-206-3147). Of the filtrate, 39\u2009\u00b5l was subjected to mass spectrometry analysis on an Agilent Technologies triple quad 6495C mass spectrometer. For HPLC (Agilent 1290 Infinity II), buffer\u2009A (described above) and buffer\u2009B (0.0075% formic acid in acetonitrile (Honeywell, LC015) were used in combination with an alkyl reversed-phase column (Zorbax RRHD StableBond Aq, 2.1\u2009\u00d7\u2009150\u2009mm, 1.8\u2009\u00b5m, 80\u2009\u00c5, Agilent Technologies, 859700-914); concentration buffer\u2009B: 0\u20131\u2009min 0%, 1\u20131.4\u2009min 0.1%, 1.4\u20132.8\u2009min 0.4%, 2.8\u20134.2\u2009min 0.9%, 4.2\u20135.6\u2009min 1.6%, 5.6\u20138\u2009min 4%, 8\u201311.5\u2009min 15%, 15\u201315.5\u2009min 50%, 15.5\u201316.5\u2009min 50%, 16.5\u201317\u2009min 0% and 17\u201318\u2009min 0%.<\/p>\n In vitro translation<\/p>\n In vitro translation assays were performed by incubating 50\u2009ng unmodified or modified luciferase mRNA for 3\u2009min at 65\u2009\u00b0C before adding 3.5\u2009\u03bcl RRL (Promega, L4960) in a final volume of 5\u2009\u03bcl at 30\u2009\u00b0C. Reactions were stopped at 20, 40, 60, 80, 180 and 360\u2009min by placing on dry ice. In vitro translation assays incorporating [35S]-L-methionine\/cysteine were performed by incubating 100\u2009ng unmodified or modified luciferase mRNA for 3\u2009min at 65\u2009\u00b0C before adding 3.5\u2009\u03bcl RRL containing an amino acid mixture (minus methionine) and 10\u2009\u03bcCi [35S]-L-methionine\/cysteine (Revvity, NEG772002MC) in a final volume of 5\u2009\u03bcl at 30\u2009\u00b0C according to the manufacturer\u2019s instructions. Reactions were stopped at 80\u2009min by adding 2\u00d7 Laemmli loading buffer. Proteins were denatured for 10\u2009min at 70\u2009\u00b0C before separation on 14% SDS\u2013polyacrylamide gels for PAGE. Radioactive signals were captured on a phosphoscreen for 72\u2009h before detection with a Typhoon molecular bioimager (GE).<\/p>\n IVT mRNA transfections<\/p>\n For NanoLuc, NanoLuc synthesized with G47A+884G mutant T7 polymerase, C=U NanoLuc and CD25 experiments in HeLa cells, cells were transfected in suspension with 2.5\u2009\u00b5g IVT mRNA per 1\u2009\u00d7\u2009106 cells using 3.75\u2009\u00b5l MessengerMax reagent (Invitrogen, LMRNA015) in OptiMEM (Thermo Fisher Scientific, 31985062) according to the manufacturer\u2019s instructions. After 5\u2009min (standard condition) or 24\u2009h (extended transfection) of exposure to the mRNA\u2013LNP complexes, the cells were washed twice with PBS, resuspended in fresh DMEM\u2013BCS and seeded separately for protein, RNA and luminescence experiments. At the indicated time points, cells were lysed directly in TRIzol reagent (100\u2009\u00b5l, Thermo Fisher Scientific, 15596026) or scraped into PBS before lysis in supplemented RIPA buffer (protease and phosphatase inhibitor). For FLuc experiments, plated HeLa cells were transfected at about 50% confluency with around 2.5\u2009\u00b5g IVT mRNA per 6-well plate with 3.75\u2009\u00b5l MessengerMax reagent in OptiMEM, as described above. Cells for RNA analysis were lysed immediately after transfection in TRIzol reagent. At 6\u2009h after transfection, cells for protein analysis were washed twice with PBS, scraped into PBS and lysed in cell culture lysis buffer (Promega, E1500) supplemented with protease and phosphatase inhibitor. To generate dsRNA-treated controls, HeLa cells were transfected with 0.5\u2009\u00b5g\u2009ml\u20131 polyI:C (Millipore Sigma, P1530) for 2\u2009h, using 12\u2009\u00b5l MessengerMax reagent and 4\u2009ml OptiMEM, according to the manufacturer\u2019s instructions. Cells were collected for protein and RNA analyses 6\u2009h after polyI:C treatment. THP-1-derived M0 macrophages, primary human MoDCs and MEFs were transfected with 1\u2009\u00b5g IVT mRNA per 1\u2009\u00d7\u2009106 live cells in suspension before plating (MoDCs and MEFs) or in seeded adherent cells (THP-1). For U-2 OS experiments, 500,000 cells were seeded 24\u2009h before transfection of 1\u2009\u03bcg RNA using 1.5\u2009\u03bcl Lipofectamine MessengerMax according to the manufacturer\u2019s protocol. Anisomycin (Sigma Aldrich, A9789-5MG) control cells were treated at 0.2\u2009\u03bcg\u2009ml\u20131. The cells were collected with supplemented RIPA buffer (protease and phosphatase inhibitor) after 1.5\u2009h. For titration experiments, RNA and MessengerMax reagent were each serial-diluted 1:10 before forming LNP complexes. Conditions were otherwise as described for HeLa cells.<\/p>\n Electroporation<\/p>\n HeLa cells (1.1\u2009\u00d7\u2009106) were resuspended in 100\u2009\u00b5l Neon NxT resuspension buffer\u2009R, and aqueous RNA (1.1\u2009\u03bcg in 10\u2009\u03bcl) was added and gently mixed. The mixture (100\u2009\u03bcl) was aspirated into a 100\u2009\u03bcl Neon NxT Tip on a respective pipette and docked into a Neon NxT Pipette Station with the tube containing 2\u2009ml buffer E100. Electroporation was performed using the HEK293 mRNA1 protocol, and the cells were immediately transferred to complete medium for plating.<\/p>\n Luminescence<\/p>\n For in vitro translation assays, reactions were diluted with 95\u2009\u03bcl of 1\u2009mg\u2009ml\u20131 BSA, then 10\u2009\u03bcl of the dilution was mixed with 40\u2009\u03bcl PBS and 50\u2009\u03bcl Nano-Glo reagent (Promega, N1120) according to the manufacturer\u2019s instructions. In NanoLuc-transfected cells, 100\u2009\u00b5l Nano-Glo assay reagent was used per 10,000 cells. For detection of FLuc luminescence, cleared lysate was diluted to correspond to 10,000 transfected cells in 10\u2009\u00b5l Cell Culture lysis buffer (Promega, E1500). Luminescence was determined by adding 100\u2009\u00b5l Luciferase assay reagent according to the manufacturer\u2019s instructions, followed by detection using a SpectraMax iD3 (Molecular Devices) instrument.<\/p>\n Protein stability<\/p>\n To inhibit the proteasome, HeLa cells transfected with IVT mRNA were cultured with 25\u2009\u00b5M MG132 (Cell Signaling Technologies, 2194S) in DMEM\u2013BCS for 6\u2009h, followed by protein lysis. To assess protein stability, 100\u2009\u00b5g\u2009ml\u20131 cycloheximide (Sigma, C7698) was added to HeLa cells 12\u2009h after transfection. Cells were collected for protein analyses at the indicated time points.<\/p>\n PKR inhibition<\/p>\n After 5\u20136\u2009days of differentiation, MoDCs were counted and pretreated with the PKR inhibitor C16 (1.5\u2009\u03bcM, 0.0075% DMSO, Sigma, 527450) for 10\u2009min. The cells were washed and transfected with 1\u2009\u03bcg RNA per 1\u2009\u00d7\u2009106 cells as\u00a0described above and treated for 6\u2009h before collection.<\/p>\n Western blot analysis<\/p>\n Cell lysates were cleared by centrifugation at full speed for 15\u2009min at 4\u2009\u00b0C, and the protein concentration was quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, A55865). For general expression analysis, equal amounts of protein (5\u201330\u2009\u03bcg) were loaded on 4\u201312% Bis-Tris NuPAGE gels (Thermo Fisher Scientific, NP0321), separated using NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, NP0001) and transferred onto 0.2\u2009\u03bcm nitrocellulose membranes from a Trans-Blot Turbo RTA Mini kit (Bio-Rad, 1704270) according to the manufacturer\u2019s instructions. For CHX-chase, an equal volume of lysate (10\u2009\u03bcl) was analysed.<\/p>\n Membranes were blocked with 5% milk in 0.05% Tween-20 TBS buffer and incubated in a solution containing 5% milk in 0.05% Tween-20 TBS buffer and the following primary antibodies: rabbit anti-eIF2\u03b1 (1:1,000, Cell Signaling Technology, 9722), rabbit anti-p-eIF2\u03b1 ((E90), 1:1,000, Abcam, ab32157), mouse anti-NanoLuc ((965808),m1:500, R&D, MAB10026), rabbit anti-TNF ((EPR22598-212), 1:1,000, Abcam, ab255275), mouse anti-\u03b2-tubulin ((D3U1W), 1:1,000, Cell Signaling Technology, 86298), rabbit anti-CD25 ((SP176), 1:300, Abcam, ab231441), mouse anti-Flag ((M2), 1:1,000, Sigma-Aldrich, F1804), mouse anti-JNK ((1A12E1), 1:1,000, Proteintech, 66210-1-Ig), rabbit anti-phospho-SAPK\/JNK ((Thr183\/Tyr185) (81E11) 1:1,000, Cell Signaling Technology, 4668S), rabbit anti-phospho-p38 MAPK (Thr180\/Tyr182) (1:1,000, Cell Signaling Technology, 9211S), rabbit anti-p38 MAPK (1:1,000, Cell Signaling Technology, 9212S), rabbit anti-TLR8 (1:1,000, Thermo Fisher Scientific, PA5-102413), rabbit anti-TLR7 ((EPR2088(2)), 1:1,000, Abcam, ab124928), rabbit anti-lamin B1 ((EPR8985(B)), 1:1,000, Abcam, ab133741), rabbit anti-ZNF598 ((5H5L17), 1:1,000, Thermo Fisher Scientific, 703601), mouse anti-PKR ((1441CT628.33.40), 1:1,000, Thermo Fisher Scientific, M5-37667), rabbit anti-phospho-PKR\/EIF2AK2-T446 ((ARC0293), 1:1,000, ABclonal, AP1134) and mouse anti-GAPDH ((6C5), 1:1,000, Santa Cruz Biotechnology, sc-32233). After overnight incubation at 4\u2009\u00b0C, membranes were washed 3\u2009times in 0.05% Tween-20 TBS buffer, followed by incubation with the horseradish-peroxidase-conjugated secondary antibodies anti-mouse IgG (1:5,000, GE Healthcare, NA931) or anti-rabbit IgG (1:10,000, Cell Signaling Technology, 7074). Western blots were visualized by enhanced chemiluminescence using ECL Western Blotting Detection Reagents (Cytiva, RPN2209), SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34580) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096). Chemiluminescence was detected using a ChemiDoc Imaging System (Bio-Rad). Densitometry was performed using ImageLab software (Bio-Rad).<\/p>\n RNase T2 treatment<\/p>\n RNA (500\u2009ng) was diluted with water. Human RNase T2 protein (sf9, His, MedChemExpress, HY-P76006) was added at the indicated concentrations in sodium acetate buffer (final 50\u2009mM, pH\u20094.5) and EDTA (final 2\u2009mM, pH\u20098.0). After incubation at 37\u2009\u00b0C for 5\u2009min, reactions were quenched with denaturing gel buffer, heated to 65\u2009\u00b0C for 5\u2009min and separated on a denaturing gel.<\/p>\n Translation imagingMicroscopy<\/p>\n Fixed-cell data were acquired on a wide-field upright Nikon Eclipse Ni microscope, which was controlled by Nikon Elements software. The system was equipped with a Spectra X LED light engine (Lumencor), an Orca 4.0 v.2 sCMOS camera (Hamamatsu) and a \u00d760 oil-immersion objective lens (1.4 NA, Nikon). The x-y pixel size was 108.3\u2009nm.<\/p>\n Live-cell data were acquired using a custom inverted Nikon Eclipse Ti-2E microscope, which was controlled by Nikon Elements software. The setup included an iLas2 Ring TIRF system with a \u00d760 apochromatic oil-immersion TIRF objective lens (1.49 NA, Nikon MRD01691), an ORCA-Fusion BT sCMOS camera (Hamamatsu) with a 6.5\u2009\u00b5m pixel size, an LUNF-XL multilaser unit with 405\u2009nm, 488\u2009nm, 561\u2009nm and 640\u2009nm lasers (50\u2009mW, 60\u2009mW, 50\u2009mW and 40\u2009mW, respectively), with a TRF89901-EMV2 ET quad-band filter set (Chroma) optimized for 405, 488, 561 and 640\u2009nm wavelengths for TIRF application. The x-y pixel size was 108.3\u2009nm.<\/p>\n Fixed-cell smFISH\u2013IF experiments<\/p>\n smFISH\u2013IF was performed following previously described protocols32<\/a>,36<\/a>. In brief, 12\u2009mm no.\u20091 German glass coverslips (Electron Microscopy Sciences, 72290-03) were carefully distributed into the wells of a 24-well tissue culture plate (Falcon, 353226) and cleaned with 3\u2009M sodium hydroxide for 5\u2009min followed by 3\u2009washes in Dulbecco\u2019s PBS (DPBS) (Corning, 21-031-CM). The coverslips were coated with a 1:400 dilution of fibronectin (Sigma-Aldrich, F1141-2 mg) in DPBS for 30\u2009min at 37\u2009\u00b0C, followed by one wash with DMEM. Next, 25,000 U-2 OS cells stably expressing the SINAPs accessory proteins OsTIR2\u2013IRES\u2013scFV\u2013sfGFP were seeded on each coverslip. One day after seeding, the medium was exchanged and supplemented with 1\u2009\u00b5M 5-phenyl-indole-3-acetic acid (Fisher Scientific, NC195789). IVT mRNA (10\u2009ng) was transfected into the cells using 1\u2009\u00b5l Lipofectamine MessengerMAX transfection reagent (Invitrogen, LMRNA003) following the manufacturer\u2019s protocol. Cells were incubated with the transfection reagent for 10\u2009min, washed 3\u2009times with prewarmed DMEM\u201310% FBS and left to incubate for an additional 1\u2009h in DMEM\u2013FBS. The cells were washed 3\u2009times with PBS supplemented with 5\u2009mM magnesium chloride (PBSM, Millipore, M2670), then subsequently fixed at room temperature for 10\u2009min in 4% paraformaldehyde (Electron Microscopy Sciences, 50-980-492) diluted in PBSM. Three 5-min washes were performed with 1\u00d7 PBSM to remove the fixation buffer. Cells were permeabilized in a buffer (PBSM\u2009+\u20095\u2009mg\u2009ml\u20131 BSA (VWR, 0332)\u2009+\u20090.1% Triton-X100 (Millipore, T8787)) at room temperature for 10\u2009min, followed by another three 5-min washes with 1\u00d7 PBSM. The cells were treated with a pre-hybridization buffer consisting of 2\u00d7 SSC (Corning, 46-020-CM), 10% formamide (Millipore Sigma, F9037) and 5\u2009mg\u2009ml\u20131 BSA for 30\u2009min. During the pre-hybridization step, the final hybridization solution was prepared: 60\u2009nM SunTag_v4-Cy5 smFISH probes, 60\u2009nM MBS_v5-Cy3 smFISH probes36<\/a>, chicken anti-GFP antibody (1:1,000, Aves Labs, GFP-1010), 100\u2009units\u2009ml\u20131 SUPERase\u00b7In (Thermo Fisher Scientific, AM2694), 1\u2009mg\u2009ml\u20131 competitor E.\u2009coli tRNA (Millipore Sigma, 10109541001), 2\u2009mM ribonucleoside vanadyl complex (NEB, S1402S), 10% formamide, 2\u00d7 SSC and 10% w\/v dextran sulfate (Millipore Sigma, D8906). The coverslips were incubated for a total of 3\u2009h in the hybridization solution at 37\u2009\u00b0C. After hybridization, the coverslips were washed 4\u2009times with a solution of 10% formamide in 2\u00d7 SSC. The coverslips were stained with a secondary antibody (Alexa-488-goat anti-chicken IgY secondary antibody, Thermo Fisher Scientific, A-11039) in a buffer (10% formamide, 2\u00d7 SSC) twice at 37\u2009\u00b0C for 20\u2009min. The cells were quickly washed 3\u2009times with 2\u00d7 SSC to remove any unbound secondary antibodies before a final 5-min wash in 2\u00d7 SSC. The coverslips were carefully removed from the culture dish and mounted with ProLong Diamond antifade reagent containing DAPI (Invitrogen, P36962) on a pre-cleaned frosted glass slide (Thermo Fisher Scientific, 12-552-3). The coverslips were left in the dark for more than 24\u2009h and sealed with clear nail polish. Fixed-cell imaging was performed on the day following each respective smFISH\u2013IF experiment.<\/p>\n smFISH\u2013IF time-course experiments with balancer mRNAs<\/p>\n Cells were transfected with a mixture of 10\u2009ng SINAPs reporter mRNA and 490\u2009ng CD25 balancer mRNA with the same nucleotide modification using 1\u2009\u00b5l Lipofectamine MessengerMAX transfection reagent (Invitrogen, LMRNA003) following the manufacturer\u2019s instructions. All experimental wells were transfected at the same time with the same master mix of reagents. Cells were incubated with the transfection mixture for 10\u2009min, after which the transfection medium was removed and cells were washed three times with pre-warmed, equilibrated culture medium, at which point the time was marked as zero (t\u2009=\u20090). At 10, 20, 30, 60, 120 and 240\u2009min after the wash, different wells were fixed and subject to the smFISH\u2013IF procedure as described above.<\/p>\n Ribosome runoff experiments<\/p>\n For live-cell imaging experiments, U-2 OS cells stably expressing OsTIR1\u2013IRES\u2013scFV\u2013sfGFP and tdMCP\u2013mScarlet\u2013tagRFPT\u2013CAAX were seeded onto 35\u2009mm glass-bottom dishes (Cellvis, D35-20-1.5-N) 48\u2009h before transfection (around 80,000 cells per dish). The medium was refreshed 24\u2009h before transfection, and 500\u2009\u00b5M 3-indoleacetic acid (Millipore Sigma, I2886) was added to degrade mature proteins and to minimize background fluorescence.<\/p>\n On the day of the experiment, each dish was transfected with 100\u2009ng IVT mRNA diluted in 125\u2009\u00b5l Opti-MEM mixed with 5\u2009\u00b5l Lipofectamine MessengerMax transfection reagent (Invitrogen, LMRNA003) diluted in 120\u2009\u00b5l Opti-MEM following the manufacturer\u2019s protocol. Cells were incubated with the transfection reagent for 1\u2009h, washed 3\u2009times with prewarmed DMEM\u2013FBS and then rinsed with FluoroBrite DMEM (Gibco, A1896701) supplemented with 10% FBS. Following 1\u2009h of incubation in imaging medium, dishes were transferred to the microscope stage for live-cell imaging. Cells were maintained at 37\u2009\u00b0C and 5% CO2 during imaging inside a Toki hit temperature-controlled stage top incubator. Positively transfected cells actively undergoing translation were identified by detecting translation signals in the GFP channel as well as positive RFP signal in the RNA channel, with each cell displaying approximately 20\u201340 tethered mRNAs.<\/p>\n Harringtonine treatment and ribosome runoff experiments were performed according to a previously established protocol32<\/a>. In brief, for each experiment, 3\u20134 positively transfected cells with active TLSs and close to each other were selected for imaging. After saving the cell positions and microscope focus, 0.75\u2009ml imaging medium was removed from the dish and mixed with harringtonine in a 1.5\u2009ml centrifuge tube, then added back to the dish, gently and thoroughly mixed to a final concentration of 9\u2009\u00b5g\u2009ml\u20131 with the original medium in the dish by pipetting. Imaging started 1\u2009min after adding harringtonine and the selected cells were imaged every 10\u2009s for 30\u2009min, with sequential laser excitation at 488\u2009nm and 561\u2009nm. Both channels were captured with a 500\u2009ms camera exposure time.<\/p>\n smFISH\u2013IF experiments with harringtonine ribosome runoffs<\/p>\n Cells were transfected with 10\u2009ng SINAPs reporter mRNA using 1\u2009\u00b5l Lipofectamine MessengerMAX transfection reagent (Invitrogen, LMRNA003), following the manufacturer\u2019s instructions. All experimental wells were transfected at the same time with the same master mix of reagents. Cells were incubated with the transfection mixture for 10\u2009min, after which the transfection medium was removed, and cells were washed 3\u2009times with prewarmed, equilibrated culture medium, at which point the time was marked as zero (t\u2009=\u20090). Harringtonine treatment was performed as described above, with the final concentration of harringtonine as 9\u2009\u00b5g\u2009ml\u20131. At 0, 3, 5 and 15\u2009min after harringtonine addition, the cells were washed 3\u2009times with PBS supplemented with 5\u2009mM magnesium chloride (PBSM, Millipore, M2670), then subsequently fixed at room temperature for 10\u2009min in 4% paraformaldehyde (Electron Microscopy Sciences, 50-980-492) diluted in PBSM. The subsequent smFISH\u2013IF procedure was performed as described above.<\/p>\n Ribosome runoff experiments after knocking down ZNF598<\/p>\n At 48\u2009h before imaging, 5\u2009pmol DsiRNA (Integrated DNA Technologies; ZNF598: hs.Ri.ZNF598.13.1-3; or control siRNA: 51-01-14-03) was diluted in 50\u2009\u00b5l Opti-MEM mixed with 1.5\u2009\u00b5l Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778). While the transfection mixture was incubated at room temperature for 10\u2009min, U-2 OS cells stably expressing OsTIR1\u2013IRES\u2013scFV\u2013sfGFP and tdMCP\u2013mScarlet\u2013tagRFPT\u2013CAAX were seeded onto 4-chamber 35\u2009mm glass-bottom dishes (Cellvis, D35C4-20-1.5-N) (30,000 cells per well). The transfection mixture was added to the dish immediately after cell seeding. Ribosome runoff was performed as described above with the following modifications: 20\u2009ng mRNA was transfected per well, and a final concentration of 9\u2009\u00b5g\u2009ml\u20131 harringtonine was added per well to initiate the runoff.<\/p>\n Image analysis and quantificationRibosome runoff<\/p>\n Single-molecule imaging analysis with a Matlab pipeline built around U-Track has been previously described36<\/a>,54<\/a>. The first step involved the detection of mRNAs and TLSs in each frame using the AirLocalize algorithm55<\/a>,56<\/a>. The second step used the U-Track software for tracking individual mRNAs and TLSs separately. The third step used a custom-built colocalization algorithm to link the detected mRNA and TLS tracks. After automatic detection, all tracks were manually inspected and verified using a custom-built TrackViewer in Matlab. To calculate ribosome off-time, we only analysed tracks with translating mRNAs at the beginning (the first five frames when harringtonine was added), and when the RNA signal persisted after the disappearance of the translation signal (for 3\u2009frames). This approach ensured that we did not count mRNAs leaving the imaging field due to untethering from the membrane. The ribosome runoff time was defined as the time point when the TLS intensity dropped below 10% of the one at t\u2009=\u20090. We calculated the Kaplan\u2013Meier survival probability with all runoff times (Fig. 4d<\/a>).<\/p>\n smFISH\u2013IF<\/p>\n Fixed-cell smFISH\u2013IF experiments were analysed using a custom-built Matlab pipeline as previously described32<\/a>. To detect RNA, we first manually segmented cell boundaries of successfully transfected cells and their DAPI-stained nuclei with FISH-Quant57<\/a>. After filtering, an intensity threshold was used to identify potential fluorescent spots in the image. The locations and the fluorescent intensities of the candidate spots were fitted using a 3D Gaussian function. The SunTag and MS2 FISH probes were labelled with two different colours and detected separately. To correct chromatic aberration, we prepared 100\u2009nm TetraSpek multicolour beads (Invitrogen, T7279) on a coverslip and imaged at the same channels as our experimental samples58<\/a>. SunTag and MS2 spots were colocalized with a nearest-neighbour analysis with a 3-pixel threshold after chromatic correction. We focused on intact mRNAs that contained both SunTag and MS2 FISH signals. To calculate the TLS intensity, we fit a 15\u2009\u00d7\u200915-pixel region around the SunTag FISH spot in the IF channel to a 3D Gaussian function. To measure the intensity of a single mature protein, a similar single-particle detection method was performed in the IF channel excluding the 15\u2009\u00d7\u200915-pixel RNA-containing region. By normalizing the total integrated intensity of the TLS by the median single-peptide intensity in the same cell, we estimated the number of ribosomes actively translating on the mRNA.<\/p>\n ZNF598\u2013HaloTag colocalization detection and track analysis<\/p>\n To quantify interactions between ZNF598\u2013HaloTag and TLSs, we developed a custom colocalization detection script in Matlab (R2021a) tailored for weak live-cell single-molecule fluorescence intensity time series. For each trace, the fluorescence intensity of ZNF598 was first smoothed using a 3-frame moving average filter to reduce frame-to-frame noise. Positive events were defined as contiguous time windows when the smoothed signal exceeded a threshold for at least 5\u2009frames, corresponding to 10\u2009s at a 2-s frame rate. The threshold was user defined (typically 1.5 times the background intensity and may be adjusted to slightly higher for traces with low signal variability). To tolerate brief signal disappearance (for example, due to molecules moving out of the evanescent field), we incorporated a merging criterion. Events separated by fewer than 3 frames were merged if the gap region exceeded 75% of the threshold or the pre-gap and post-gap intensities were similar (normalized difference less than 20%). The colocalization detection events were saved in a logical mask variable for each trace. These variables were subsequently used to compute interaction durations, ZNF598\u2013HaloTag intensities and the fraction of colocalization. The detection was visually validated on more than 50 randomly selected traces per condition to ensure accuracy and robustness across replicate datasets. We confirmed correct colocalization start and end points and verified that the script appropriately merged or excluded brief subthreshold gaps.<\/p>\n Mouse studies<\/p>\n DLin-MC3-DMA was purchased from MedKoo Biosciences. 18PG and DMG-PEG-2000 were obtained from Avanti Polar Lipids. Cholesterol was from Sigma-Aldrich.<\/p>\n LNP synthesis and characterization<\/p>\n Parameters for LNP synthesis were as previously described24<\/a>,25<\/a>,26<\/a>. In brief, to prepare the organic phase, a mixture of DLin-MC3 DMA, cholesterol, DMG-PEG2000 and 18PG was dissolved in ethanol. To prepare the aqueous phase, corresponding mRNA was prepared in magnesium acetate buffer (25\u2009mM, pH\u00a04.0, Fisher Scientific, SB85-1). All mRNA samples were stored at \u221280\u2009\u00b0C and thawed on ice before use. For LNP synthesis, the aqueous and ethanol phases prepared were mixed at a 3:1 ratio in a flash nanocomplexation device using syringe pumps, purified by dialysis against deionized water using a 100-kDa molecular weight cutoff cassette (Thermo Fisher Scientific) at 4\u2009\u00b0C for 24\u2009h and stored at 4\u2009\u00b0C before injection. The size, polydispersity index and zeta potentials of LNPs were measured using dynamic light scattering (ZetaPALS, Brookhaven Instruments). Diameters are reported as the intensity mean average.<\/p>\n Characterization of the encapsulation efficiency of mRNA LNPs<\/p>\n The encapsulation efficiency of mRNA in LNPs was evaluated using a Quant-iT RiboGreen assay (Thermo Fisher Scientific, R11490). To disrupt the LNP structure and release the encapsulated mRNA, LNP samples were treated with 0.5% w\/v Triton X-100 (Sigma-Aldrich, T8787). Both Triton-treated and untreated LNP samples were diluted to a concentration of less than 1\u2009\u03bcg mRNA per ml before being mixed with an equal volume of RiboGreen working solution (200-fold dilution). Standard curves were prepared using free mRNA solutions with or without 0.5% w\/v Triton X-100, covering a concentration range of 0.1\u20131.0\u2009\u03bcg mRNA per ml. Fluorescence measurements (excitation of 480\u2009nm, emission of 520\u2009nm) were recorded, and the concentrations of free mRNA (untreated samples) and total mRNA (Triton-treated samples) in the LNP formulations were quantified by comparing the fluorescence intensities against the corresponding standard curves59<\/a>.<\/p>\n Animals<\/p>\n All animal procedures were performed with ethical compliance and approval by the Johns Hopkins Institutional Animal Care and Use Committee (protocol no. MO23E31). Female BALB\/c mice (6\u20138\u2009weeks) were obtained from the Jackson Laboratory and randomly grouped. Mice were generally fed a diet containing low fibre (5%), protein (20%) and fat (5\u201310%). The pelleted feed was supplied. Mice were supplied feed free choice and they ate 4\u20135\u2009g a day (12\u2009g per 100\u2009g body weight per day). Water was supplied free choice and they usually drank 3\u20135\u2009ml a day (1.5\u2009ml per 10\u2009g body weight per day). Water was supplied using automatic waterers. Mouse rooms were maintained at 30\u201370% relative humidity and a temperature of 18\u201326\u2009\u00b0C (64\u201379\u2009\u00b0F) with at least 10 room air changes per hour. The mice were housed in standard shoebox cages with filter tops under standard specific pathogen-free conditions with a 12-h light\u2013dark cycle. Mice were provided with corncob as bedding. Sample sizes were selected based on our previous published work using similar mRNA\u2013LNPs in vivo immune profiling and biodistribution studies25<\/a>, for which comparable group sizes were sufficient to detect biologically meaningful differences. The in vivo IVIS experiments were independently replicated twice, and the datasets were pooled for analyses. Mice were randomly allocated into experimental groups. In vivo experiments and data collection were performed blinded to group allocation. Investigators remained blinded during data analysis whenever feasible.<\/p>\n The LNPs were intravenously injected into mice via the lateral tail vein at a predetermined dose per mouse. The mice were intraperitoneally injected with 100\u2009\u03bcl of 30\u2009mg\u2009ml\u20131 Nano-Glo Fluorofurimazine In Vivo substrate (FFz, Promega, N4100) solution and were anaesthetized in a ventilated anaesthesia chamber with 1.5% isoflurane in oxygen and imaged at 5\u2009min after the injection with an in vivo imaging system (IVIS, Perkin-Elmer). Luminescence was quantified using Living Image software (Perkin-Elmer).<\/p>\n Sucrose density centrifugation<\/p>\n At 1.5\u2009h after transfection, HeLa cells transfected with IVT mRNA were washed with ice-cold PBS and scraped into lysis buffer (50\u2009mM HEPES pH\u20097.4, 100\u2009mM potassium acetate, 15\u2009mM magnesium acetate, 1\u2009mM DTT, 1% Triton X-100 and emetine (360\u2009\u00b5M, Sigma, 324693-250MG)) on ice. Lysates were incubated at 4\u2009\u00b0C for 10\u2009min before clearing for 10\u2009min at 4\u2009\u00b0C and 10,000g. RNA concentration was determined using a Qubit\u20094 Fluorometer (Thermo Fisher Scientific) and about 60\u2009\u00b5g was diluted to a total volume of 600\u2009\u00b5l with lysis buffer before layering on top of 10\u201345% sucrose gradients (25\u2009mM HEPES pH\u20097.4, 100\u2009mM potassium acetate, 5\u2009mM magnesium acetate and 1\u2009mM DTT) prepared with a Biocomp Gradient Master. Centrifugation to separate ribosome fractions was performed for 1\u2009h and 45\u2009min at 41,000\u2009rpm (SW41Ti, Optima XPN-80, Beckman Coulter) at 4\u2009\u00b0C. Gradients were fractionated, and UV (A260) absorbance across the gradients was measured using a top-down Biocomp Piston Gradient Fractionator with a Triax flow cell per the manufacturer\u2019s instructions. To each fraction, 2\u2009volumes of 100% ice-cold ethanol was added and the RNA was precipitated at \u221280\u2009\u00b0C. RNA was isolated by pelleting for 30\u2009min at 18,500g and 4\u2009\u00b0C, followed by resuspension in LET buffer (25\u2009mM Tris pH\u20098.0, 100\u2009mM LiCL and 20\u2009mM EDTA), addition of 1% SDS and double acid phenol\u2013chloroform\u2013LET extraction. Supernatants containing RNA were isolated after ammonium acetate\u2013ethanol precipitation containing 1\u2009\u00b5l GlycoBlue Coprecipitant (Thermo Fisher Scientific, AM9516) per sample.<\/p>\n RNA isolation and RT\u2013qPCR<\/p>\n Unless described otherwise, total RNA was prepared by lysing cells at the indicated time points in TRIzol (100\u2009\u00b5l reagent per 1\u20132\u2009\u00d7\u2009105 cells) or adding an equal volume to in vitro lysates and extracted according to the manufacturer\u2019s instructions. RNA was reverse transcribed with oligo(dT) primers or random hexamers using a Superscript IV system (Thermo Fisher Scientific, 18090200) according to the manufacturer\u2019s suggestions, followed by qPCR with specific primers (Supplementary Table 1<\/a>) using LightCycler 480 SYBR Green I master mix (Roche, 04887352001) in a LightCycler 96 Instrument (Roche).<\/p>\n RNA-seq library preparation and analysis<\/p>\n In brief, 1\u2009\u00b5g modified and unmodified mRNA of the +1 FS FLuc reporter was used to generate cDNA libraries with a NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEB, E7760) starting from the fragmentation step. NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, NEB, E6440) were used with 6\u2009cycles of PCR amplification. The concentrations of the indexed libraries were analysed on an Agilent 4200 TapeStation (Agilent Technologies) using a D1000 kit (Agilent Technologies). Equimolar amounts of the indexed libraries were pooled to obtain a 2\u2009nM library mixture. After further dilution, the final 750\u2009pM library was sequenced single-end with dual index (122\u2009\u00d7\u20098\u2009\u00d7\u20098) using Illumina NextSeq2000 P1 reagents (100 cycles) on an Illumina NextSeq2000 instrument following the manufacturer\u2019s instructions (Illumina).<\/p>\n RNA-seq analysis<\/p>\n The quality of raw reads was assessed using FastQC (v.0.12.1)60<\/a>. The average per-base quality score across all the files was greater than 32, and contamination of Illumina adapters was observed at the 3\u2032 end of the reads. These adapters were removed using Trim Galore (v.0.6.11)61<\/a>. As the sequenced RNA was from a construct with a known open reading frame, the libraries (both raw and trimmed) were aligned using bowtie2 (v.2.5.3)62<\/a>,63<\/a>. The average alignment rate of the raw reads was 94% and increased to 99.52% for the trimmed reads. The aligned reads were sorted, indexed and summary statistics (flagstat) were obtained using samtools (v.1.23)64<\/a>. The mapped sequences with MAPQ\u2009>\u2009=\u20095 were used to calculate the insertion and deletion rate in terms of per nucleotide using Qualimap (v.2.2.1)65<\/a> across the open reading frame. The libraries were visualized using IGV66<\/a>,67<\/a>.<\/p>\n Ribo-seq sample and library preparation<\/p>\n Hela cells were grown to 70\u201380% confluency before co-transfection with 3\u2009\u00b5g mRNA for the WT 5\u00d7U FLuc reporter per 10\u2009cm dish for each modification (unmodified, ac4C and m1\u03a8) using MessengerMax reagent (22.5\u2009\u03bcl per dish). Three 10\u2009cm2 plates were transfected for each modification. After 3\u2009h, the cells were washed with PBS and the samples were collected on wet ice in 500\u2009\u00b5l of lysis buffer (see the section \u2018Sucrose density centrifugation\u2019 with inclusion of complete mini protease inhibitor cocktail, Roche, 11 836 153 001). Cell lysates were next triturated 10 times and clarified by centrifugation at 20,000g for 10\u2009min at 4\u2009\u00b0C.<\/p>\n Ribosome profiling and RPF purification was performed according to a published protocol68<\/a> but with modifications. In brief, for ribosome profiling, micrococcal nuclease\u2009I (MNase\u2009I, 2,400\u2009U) was used to digest clear lysate equivalent to an optical density of 3.5 at room temperature for 40\u2009min. MNase\u2009I was used in place of the more commonly used RNase\u2009I because both ac4C and m1\u03a8 confer resistance to RNase\u2009I cleavage69<\/a>,70<\/a>, which would have introduced systematic bias against modified transcripts and precluded direct comparison of ribosome footprint densities across modification states. Reactions were stopped using 10\u2009mM EGTA and 5\u2009\u03bcl SUPERaseIn. Digested lysates of unmodified, ac4C-modificed and m1\u03a8-modified samples were loaded onto 10\u201345% sucrose gradients as described above for 1\u2009h and 40\u2009min. The monosome fraction was collected and RNA was isolated from ribosome-protected fragments using the phenol\u2013chloroform\u2013LET method (described in the section \u2018Sucrose density centrifugation\u2019). Next, 25\u201335\u2009bp size RNA was purified from 15% urea\u2013PAGE using gel extraction buffer (300\u2009mM sodium acetate (pH\u20095.5), 1\u2009mM EDTA and 0.25% (w\/v) SDS). Library preparation was performed using a QiaSeq miRNA Library kit (Qiagen, 331502) and sequenced on a miSeq Illumina platform.<\/p>\n Ribo-seq analysis<\/p>\n Raw sequencing reads were processed to remove adapter sequences and to extract unique molecular identifiers (UMIs) using UMI-tools (v.1.1.5)71<\/a> with the following settings: –extract method=regex \u2013bc pattern=\u2018.+(?PAACTGTAGGCACCATCAAT){s<=2} (?P.{12})(?P.+)\u2019. UMI-extracted reads were mapped to the FLuc sequence using Bowtie (v.1.3.1)72<\/a> with the following settings: -a -v 2 \u2013norc. Extracted UMIs and mapping coordinates were used to remove duplicate reads using the dedup function from UMI-tools with the following options: –method unique –extract-umi-method=read_id –umi-separator=\u201c_\u201c.<\/p>\n