Dominant roles of BRCA1 in cellular tolerance to a chain-terminating nucleoside analog, alovudine.

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.

Short Activators and Repressors of RNA Toehold Switches.

RNA toehold switches are a widely used class of molecule to detect specific RNA "trigger" sequences, but their design, intended function, and characterization to date leave it unclear whether they can function properly with triggers shorter than 36 nucleotides. Here, we explore the feasibility of using standard toehold switches with 23-nucleotide truncated triggers. We assess the crosstalk of different triggers with significant homology and identify a highly sensitive trigger region where just one mutation from the consensus trigger sequence can reduce switch activation by 98.6%. However, we also find that triggers with as many as seven mutations outside of this region can still lead to 5-fold induction of the switch. We also present a new approach using 18- to 22-nucleotide triggers as translational repressors for toehold switches and assess the off-target regulation for this strategy as well. The development and characterization of these strategies could help enable applications like microRNA sensors, where well-characterized crosstalk between sensors and detection of short target sequences are critical.

A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes.

Seasonal influenza vaccines offer little protection against pandemic influenza virus strains. It is difficult to create effective prepandemic vaccines because it is uncertain which influenza virus subtype will cause the next pandemic. In this work, we developed a nucleoside-modified messenger RNA (mRNA)-lipid nanoparticle vaccine encoding hemagglutinin antigens from all 20 known influenza A virus subtypes and influenza B virus lineages. This multivalent vaccine elicited high levels of cross-reactive and subtype-specific antibodies in mice and ferrets that reacted to all 20 encoded antigens. Vaccination protected mice and ferrets challenged with matched and mismatched viral strains, and this protection was at least partially dependent on antibodies. Our studies indicate that mRNA vaccines can provide protection against antigenically variable viruses by simultaneously inducing antibodies against multiple antigens.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

Export of RNA-derived modified nucleosides by equilibrative nucleoside transporters defines the magnitude of autophagy response and Zika virus replication.

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

Adaptor Molecules Epitranscriptome Reprograms Bacterial Pathogenicity.

The strong decoration of tRNAs with post-transcriptional modifications provides an unprecedented adaptability of this class of non-coding RNAs leading to the regulation of bacterial growth and pathogenicity. Accumulating data indicate that tRNA post-transcriptional modifications possess a central role in both the formation of bacterial cell wall and the modulation of transcription and translation fidelity, but also in the expression of virulence factors. Evolutionary conserved modifications in tRNA nucleosides ensure the proper folding and stability redounding to a totally functional molecule. However, environmental factors including stress conditions can cause various alterations in tRNA modifications, disturbing the pathogen homeostasis. Post-transcriptional modifications adjacent to the anticodon stem-loop, for instance, have been tightly linked to bacterial infectivity. Currently, advances in high throughput methodologies have facilitated the identification and functional investigation of such tRNA modifications offering a broader pool of putative alternative molecular targets and therapeutic avenues against bacterial infections. Herein, we focus on tRNA epitranscriptome shaping regarding modifications with a key role in bacterial infectivity including opportunistic pathogens of the human microbiome.

Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain.

We report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster.

Pseudouridimycin (PUM) was recently discovered from Streptomyces sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of Streptococcus pyogenes peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host Streptomyces coelicolor M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC.

Nucleoside selectivity of Aspergillus fumigatus nucleoside-diphosphate kinase.

Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s-1 ), specificity constant (626 ± 110.0 mm-1 ·s-1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol-1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s-1 ) and lowest specificity constant (40.2 ± 4.4 mm-1 ·s-1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV).

Effects of fungus-host associations on nucleoside differences among Ophiocordyceps sinensis populations on the Qinghai-Tibet Plateau of China.

Nucleosides are active components in Ophiocordyceps sinensis. All individual nucleoside contents in both fruiting and caterpillar bodies were significantly different among the 24 O. sinensis populations that are distributed across the Qinghai-Tibet Plateau of China. The nucleoside contents of the fruiting and caterpillar bodies show significant positive correlations with the geographical distances of O. sinensis populations, and the nucleoside contents in the O. sinensis populations with the non-dominant fungus-host haplotype associations from isolated geographical areas show significant positive correlations with the genetic distances among those populations based on nrDNA ITS and COI sequences, respectively. The fungus-host associations play important roles in the nucleoside differences of O. sinensis populations.

Combination of c oxidase subunit I based deoxyribonucleic acid barcoding and HPLC techniques for the identification and quality evaluation of Pheretima aspergillum.

This study aimed to identify Pheretima aspergillum (Guang-Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high-performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura-2-Parameter model was used to analyze genetic distance, and phylogenetic neighbor-joining tree was constructed for species identification of 20 labeled Guang-Pheretima samples. A high-performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang-Pheretima (P. aspergillum), and the others as the adulterants-Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor-joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant-M. magna. The incidence of adulterants for Guang-Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant- or animal-derived products for safety concerns to avoid misidentification.

Specificity in the biosynthesis of the universal tRNA nucleoside N6-threonylcarbamoyl adenosine (t6A)-TsaD is the gatekeeper.

N6-threonylcarbamoyl adenosine (t6A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t6A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO2, and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A37 of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn6A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t6A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t6A pathway, we carried out biochemical and biophysical investigations of the t6A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn6A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn6A in their tRNA, we conclude that their origin is due to formation via the t6A pathway.

Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS-PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside.

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion.

The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3' end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.We provide a step-by-step instruction for time-resolved mRNA 3' end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.

The Amipurimycin and Miharamycin Biosynthetic Gene Clusters: Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucleoside Antibiotics.

Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (-)-cispentacin and N5-hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny. Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimycin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the biosynthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which, along with labeled precursor feeding studies, implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N5-hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (-)-cispentacin.

Direct Prebiotic Pathway to DNA Nucleosides.

It is assumed that RNA played a key role in the origin of life, and the transition to more complex but more stable DNA for continuous information storage and replication requires the development of a ribonucleotide reductase to obtain the deoxyribonucleotides from ribonucleotides. This step, as well as an alternative path from abiotic molecules to DNA-based life is completely unknown. Shown here is the formation of deoxyribonucleosides under relevant prebiotic conditions in water in high regio- and stereoselectivity, from all canonical purine and pyrimidine bases, by condensation with acetaldehyde and sugar-forming precursors. Thus, a continuous path to deoxyribonucleosides, starting from simple, prebiotically available molecules has been discovered. Furthermore, the deoxyapionucleosides (DApiNA) were identified as a potential DNA progenitor. The results suggest that the DNA world evolved much earlier than previously assumed.

DNA Adduct-Directed Synthetic Nucleosides.

Chemical damage to DNA is a key initiator of adverse biological consequences due to disruption of the faithful reading of the genetic code. For example, O6-alkylguanine ( O6-alkylG) DNA adducts are strongly miscoding during DNA replication when the damaged nucleobase is a template for polymerase-mediated translesion DNA synthesis. Thus, mutations derived from O6-alkylG adducts can have severe adverse effects on protein translation and function and are an early event in the initiation of carcinogenesis. However, the low abundance of these adducts places significant limitations on our ability to relate their presence and biological influences with resultant mutations or disease risk. As a consequence, there is a critical need for novel tools to detect and study the biological role of alkylation adducts. Incorporating DNA bases with altered structures that are derived synthetically is a strategy that has been used widely to interrogate biological processes involving DNA. Such synthetic nucleosides have contributed to our understanding of DNA structure, DNA polymerase (Pol) and repair enzyme function, and to the expansion of the genetic alphabet. This Account describes our efforts toward creating and applying synthetic nucleosides directed at DNA adducts. We synthesized a variety of nucleosides with altered base structures that complement the altered hydrogen bonding capacity and hydrophilicity of O6-alkylG adducts. The heterocyclic perimidinone-derived nucleoside Per was the first of such adduct-directed synthetic nucleosides; it specifically stabilized O6-benzylguanine ( O6-BnG) in a DNA duplex. Structural variants of Per were used to determine hydrogen bonding and base-stacking contributions to DNA duplex stability in templates containing O6-BnG as well as O6-methylguanine ( O6-MeG) adducts. We created synthetic probes able to stabilize damaged over undamaged templates and established how altered hydrogen bonding or base-stacking properties impact DNA duplex stability as a function of adduct structures. This knowledge was then applied to devise a hybridization-based detection strategy involving gold nanoparticles that distinguish damaged from undamaged DNA by colorimetric changes. Furthermore, synthetic nucleosides were used as mechanistic tools to understand chemical determinants such as hydrogen bonding, π-stacking, and size and shape deviations that impact the efficiency and fidelity of DNA adduct bypass by DNA Pols. Finally, we reported the first example of amplifying alkylated DNA, accomplished by combining an engineered polymerase and synthetic triphosphate for which incorporation is templated by a DNA adduct. The presence of the synthetic nucleoside in amplicons could serve as a marker for the presence and location of DNA damage at low levels in DNA strands. Adduct-directed synthetic nucleosides have opened new concepts to interrogate the levels, locations, and biological influences of DNA alkylation.

C-Nucleoside Formation in the Biosynthesis of the Antifungal Malayamycin A.

Malayamycin A is an unusual bicyclic C-nucleoside, with interesting antiviral, antifungal, and anticancer bioactivity. We report here the discovery and characterization of the biosynthetic pathway to malayamycin by using genome mining of near-identical clusters both from the known producer Streptomyces malaysiensis and from Streptomyces chromofuscus. The key precursor 5'-pseudouridine monophosphate (5'-Ψ-MP) is supplied chiefly through the action of MalD, a TruD-like pseudouridine synthase. In vitro assays showed that MalO is an enoylpyruvyltransferase acting almost exclusively on 5'-Ψ-MP rather than 5'-UMP, while in contrast the counterpart enzyme NikO in the nikkomycin pathway readily accepts either substrate. As a result, deletion of malD in S. chromofuscus coupled with introduction of the gene for NikO led to production of non-natural N-malayamycin, as well as malayamycin A. Conversely, cloning malO into the nikkomycin producer Streptomyces tendae in place of nikO diverted biosynthesis toward C-nucleoside formation.

Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance.

Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.

mRNA-Based Genetic Reprogramming.

The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient's own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several "non-integrating" reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously "footprint-free," most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic.