Around 20% of all malignancies harbour activating mutations in RAS isoforms. Despite this, there is a deficiency of RAS-targeting agents licensed for therapeutic use. The picomolar affinity of RAS for GTP, and the lack of suitable pockets for high-affinity small-molecule binding, precluded effective therapies despite decades of research. Recently, characterisation of the biochemical properties of KRAS-G12C along with discovery of its ‘switch-II pocket’ have allowed development of effective mutant-specific inhibitors. Currently seven KRAS-G12C inhibitors are in clinical trials and sotorasib has become the first one to be granted FDA approval. Here, we discuss historical efforts to target RAS directly and approaches to target RAS effector signalling, including combinations that overcome limitations of single-agent targeting. We also review pre-clinical and clinical evidence for the efficacy of KRAS-G12C inhibitor monotherapy followed by an illustration of combination therapies designed to overcome primary resistance and extend durability of response. Finally, we briefly discuss novel approaches to targeting non-G12C mutant isoforms. Mutations in RAS genes occur frequently in solid and haematological malignancies with around 20% of all tumours harbouring a mutation in at least one isoform ,2. Lung Most oncogenic mutations in RAS isoforms are missense gain-of-function mutations in codons G12, G13 or Q61 . DiffereRAS proteins are small GTPases that cycle between GDP-bound (inactive) and GTP-bound (active) conformations. GTP-RAS activates several downstream signalling cascades, the most well studied of which are the MAPK (RAF/MEK/ERK) and PI3K (PI3K/AKT/mTOR) pathways. Impaired GTP hydrolysis, resulting in increased flux through downstream pathways, is a key property of oncogenic RAS ,12. HoweIn this review, we discuss initial approaches to target oncogenic RAS, including farnesyl transferase inhibitors and small molecule inhibitors of RAS–effector pathways. We next look at the development of KRAS-G12C inhibitors, a class of molecule in advanced-phase clinical trials. Finally, we briefly discuss emerging approaches in the field.Early attempts to develop direct RAS inhibitors failed due to the picomolar affinity of RAS for GTP and the lack of suitable hydrophobic pockets which could allow high-affinity binding of small molecules . In the Due to the inherent difficulties in directly targeting RAS or its post-translational modifications, in recent years the most commonly pursued strategy has been to target RAS effector signalling. This has resulted in the development and clinical evaluation of a battery of highly selective inhibitors against downstream effectors of RAS, predominantly the RAF/MEK/ERK and PI3K/AKT/mTOR pathways. The importance of the RAF/MEK/ERK pathway for the survival and proliferation of tumour cells harbouring RAS mutations has been demonstrated in several studies ,26,27. LEven more complex has been the study of RAF inhibitors. Surprisingly, in contrast to the efficacy observed in BRAF-mutant melanoma, BRAF inhibitors were found to activate ERK and stimulate growth of RAS-mutant cancers by an effect known as the RAF inhibitor paradox ,38. ThisMore recent has been the development of ERK inhibitors ,44. HoweThe lack of clinical benefit of the existing RAF, MEK and ERK inhibitors as monotherapies could be explained by two main reasons. RAS has multiple downstream effector pathways. Therefore, targeting only MAPK signalling may not be enough for clinical efficacy. On the other hand, inhibition of ERK results in the loss of the negative feedback loops that regulate the activity of the pathway. This causes the reactivation of the MAPK pathway, and in some cases of the PI3K/AKT pathway, mainly due to upregulation of receptor tyrosine kinase (RTK) activity. This suggests that combination therapies with RAF, MEK or ERK inhibitors are needed to achieve an effective response. However, identification of optimal combinations depends not only on the biology of RAS-mutant tumours, but also on the selection of combinations that achieve a strong pathway inhibition with an adequate therapeutic index.One strategy to achieve potent MAPK inhibition and overcome resistance mechanisms is by vertical inhibition of the RAF/MEK/ERK pathway. This is now extensively used in BRAF-mutant melanoma . For exaAlthough targeting RTK-driven pathway reactivation is an attractive option, the fact that different, or even multiple, RTKs can be activated after MEK inhibition precludes the selection of a universal combination strategy. An alternative that has been recently proposed is the targeting of the RTK-associated phosphatase SHP2 (encoded by PTPN11). Although the exact biological function of SHP2 is not clear, it is known that SHP2 acts as key node linking RTK–RAS signalling, probably acting as a scaffold protein which binds GRB2 and SOS1, therefore promoting the activation of RAS proteins by stimulating GDP for GTP exchange ,64. AblaL or genetic ablation of BCL-2 [L inhibitor navitoclax is ongoing (NCT02079740). Several studies have also indicated that after MEK inhibition, KRAS-mutant tumours are more dependent on autophagy and that co-targeting MEK and autophagy results in a synergistic response [Finally, other strategies designed to improve the efficacy of MEK inhibitors involve targeting proteins that are outside the RAS pathway. Targeting the cyclin-dependent kinase 4 (CDK4) was shown to be synthetically lethal in KRAS-mutant NSCLC . These dof BCL-2 . Based oresponse ,76. One As discussed above, G12C-mutant KRAS can be distinguished by a unique biochemical property—its relatively preserved intrinsic GTP-hydrolysis rate. In addition, the mutant cysteine renders the molecule susceptible to specific, and irreversible, binding with cysteine-reactive small molecules. These properties were exploited by Shokat and colleagues who screened 480 tethering compounds against KRAS-G12C . They diContinuing to exploit P2 for drug binding, ARS-853, a compound with robust cellular activity, was developed . ModelliTreatment of KRAS-G12C-mutant cell lines with ARS-853 resulted in pathway inhibition downstream of KRAS. However, there was heterogeneity in the depth and durability of effect across different cell lines, suggesting that factors beyond the G12C mutation may modify sensitivity. In terms of viability, some lines were insensitive to the drug, and the same cells showed lack of response to genetic ablation of KRAS, suggesting KRAS independence in spite of the G12C mutation. In 3D culture, however, all lines showed robust inhibition of proliferation following treatment , demonstThere are several active and recently completed clinical trials involving KRAS-G12C inhibitors . Of thes50 values between cell lines. MRTX849 too was able to inhibit in vitro RAS function and G12C-mutant cell viability with IC50s in the nanomolar range [1/2 = 20–24 h). Tumour regression also occurred. This was dose-dependent, with an increased proportion of complete regressions at a higher dose, possibly correlating with the degree of p-ERK inhibition [In vitro treatment with AMG 510 resulted in inhibition of phospho-ERK (p-ERK) and phospho-S6 (p-S6) and increased cleaved caspase-3 specifically in KRAS-G12C-mutant cells . Again, ar range . Pre-clihibition . Similarhibition .Following these promising pre-clinical results, early-phase clinical evidence has also been encouraging. The KRYSTAL-1 study NCT03785249) is a phase I/II study which is recruiting patients with pre-treated NSCLC, CRC and other advanced solid tumours. Arms include MRTX849 monotherapy as well as combinations with pembrolizumab, cetuximab and afatinib ,83. In o249 is a KRAS mutations in NSCLC often co-occur with mutations in tumour-suppressor genes including TP53, STK11 and KEAP1 . An explSeparately, the CodeBreak 100 trial is a large study with several phase I/II arms investigating AMG 510 as monotherapy or in combination with anti PD-1/PD-L1 therapy (NCT03600883). The phase I monotherapy portion of CodeBreak 100 treated 129 patients of whom 59 had NSCLC, 42 CRC and 28 had other tumour types ,92,93. PEarly results from the phase II portion of the AMG 510 monotherapy arm of CodeBreak 100, treating only patients with advanced NSCLC, were recently reported . Of 123 Two other G12C inhibitor trials opened for recruitment pre-2020: JNJ-74699157 and LY349934. The latter was terminated due to unexpected toxicity. This highlights the fact that despite the mutant-specific nature of inhibitors, toxicity can still occur, perhaps due to off-target effects on other cysteine-containing proteins despite reassuring in vitro specificity. Newer inhibitors are also in early-phase testing. D-1533, GDC-6036 and JDQ443 trials are all recruiting. Yet, other inhibitors are being explored pre-clinically, including inhibitors from Boehringer Ingelheim and Revolution Medicines. In contrast with the similar approach used by most of the companies, Revolution Medicines has used a completely different strategy to covalently inhibit KRAS-G12C in the active state (GTP bound). These molecules are covalent tri-complex inhibitors where the compound mediates the binding of KRAS-G12C with the chaperone cyclophilin A, preventing the binding of RAS effector proteins ,99.As with other targeted therapies, resistance to KRAS-G12C inhibitor monotherapy will limit clinical efficacy and efforts are underway to understand resistance mechanisms. The development of G12C inhibitor combinations has benefited from prior knowledge of MEK inhibitor combinations. However, with a broader therapeutic window, it is likely that the mutant-specificity of G12C inhibition will enhance synergy and mitigate toxicity compared to MEK inhibitor combinations.50 values were similar across lines, suggesting that even resistant lines undergo robust target engagement [As alluded to previously, sensitivities of cell lines to KRAS-G12C inhibition vary up to 100-fold in vitro . These sgagement . Inhibitgagement . This suIn order to understand KRAS dependency, one study looked at signalling in pancreatic cell lines following genetic ablation of KRAS. This was tolerated in a subset and while these did not display increased YAP1 activity or sensitivity, as had previously been shown in KRAS-deficient models , they diWhile combined PI3K and KRAS-G12C inhibition appears broadly effective, the studies above also suggest RTK co-inhibition could work, although they highlight the heterogeneity of optimal synergy between different cell lines. To overcome this, SHP2 inhibitors have been proposed as a way to delay adaptive resistance to G12C inhibitors . In one Although heterogeneity of RTK activation may preclude the use of specific RTK inhibitors as a general synergistic mechanism, some tumours may display characteristics suggestive of the most likely kinase to mediate resistance. For example, EGFR signalling was found to be the dominant mechanism of resistance in one study on colorectal cancer . Indeed,The studies above focussed on targeting either upstream or downstream of RAS. A third approach entails simultaneous targeting of both . UtilisiBeyond the RTK–RAS–MAPK/PI3K pathways, CRISPR and similar screens have the potential to identify novel synergistic partners. In a focussed CRISPR screen of 400 genes in cells treated with MRTX849 treatment , classicCombination strategies to prolong sensitivity to KRAS-G12C inhibitors are already in early-phase clinical trials . SeveralAlthough clinical results with KRAS-G12C inhibitors are promising, drugs targeting oncogenic drivers usually result in a rapid development of resistance after promising initial responses. On the other hand, checkpoint inhibitor therapy, which is the standard-of-care treatment for patients with advanced NSCLC who lack a driver mutation , resultsKRAS-G12C inhibition has been shown to have effects on tumour-intrinsic mechanisms of immune subversion . In five+ cross-presenting dendritic cells and macrophage infiltration after just 4 days of treatment. MEK inhibitor treatment in the same model did not have this effect and furthermore, MEK inhibition but not AMG 510 was shown to impair T-cell proliferation in a co-culture system in vitro. This may partly explain the lack of clinical success when combining MEK or BRAF inhibition with immunotherapy, while also suggesting that use of mutant-specific inhibitors such as AMG 510 could overcome this.In a separate study, long-term cures induced by AMG 510 in a subcutaneous CT26 model were also suggested to rely on anti-tumour immunity . Similar+ cells which displayed PD-1 upregulation, especially when in close proximity to tumour cells, suggesting tumour-driven activation. While these studies were performed in lung cancer, similar effects were seen in a mouse model of KrasG12C/Tp53R172H pancreatic cancer. Treatment with ARS-1620 increased the percentage of T cells while decreasing granulocytic MDSCs [Further evidence for the potential of KRAS-G12C inhibition to reduce the immunosuppressive tumour microenvironment produced by oncogenic KRAS and potentially sensitise tumours to immune checkpoint blockade came from another study using the 3LL ΔNRAS murine lung carcinoma line . Using iic MDSCs . KRAS-G1ic MDSCs . These cic MDSCs . In mousic MDSCs . Interplic MDSCs .Based on this pre-clinical evidence, trials combining KRAS-G12C inhibition and immunotherapy are underway . The CodMULTI(ON) inhibitor, active against tumours harbouring KRAS-G12D and KRAS-G12V mutations, although their portfolio also includes KRAS-G12D- and KRAS-G13C-selective inhibitors [Although the development of KRAS-G12C inhibitors is a great advance for the treatment of patients carrying this mutation, there is still a need to target other KRAS mutations. However, other approaches will be needed, as these proteins will lack the presence of the reactive cysteine that allows the covalent binding of the inhibitor. Mirati Therapeutics has developed a selective G12D inhibitor (MRTX1133) that targets both active and inactive states, although the details of the mechanism have still not been published . A cyclihibitors .Due to the wide-range of potential targets, small-interfering RNAs have promising therapeutic potential as mutant-specific RAS inhibitors. Although their short serum and intra-cellular half-life can limit efficacy, local administration may help concentrate siRNA in the tumour microenvironment. Such an approach is employed in an ongoing phase II trial (NCT01676259), where patients with pancreatic cancer receive intratumoural injection of a KRAS-G12D-specific siRNA in a biodegradable matrix  in combination with systemic chemotherapy .+ TILs resulted in regression of all seven lung lesions [*08:02 loss, suggesting a specific mechanism of escape to evade ACT-driven immunity. Another approach involves engineering T cells with RAS-specific T-cell receptor (TCR) clonotypes. As a proof-of-concept, serum from patients with NSCLC was screened for neoantigen-reactive T-cell clones. [+ reactivity from blood (but not tumour) to the KRAS-G12V-mutant allele. Donor lymphocytes were transduced with the relevant TCR clones and the engineered cells demonstrated much higher affinity for KRAS-G12V-pulsed target cells relative to those pulsed with wild-type KRAS. At the very least, these studies provide proof-of-concept that mutant RAS alleles can be immunogenic in humans and suggest the potential for subclinical anti-RAS immune responses to be augmented with ACT.Mutant-specific targeting is also possible through the use of adoptive cellular therapy (ACT). One approach involves extraction of tumour-infiltrating lymphocytes (TILs) followed by selection, expansion and infusion back into the patient. This was used in a patient with metastatic colorectal cancer, where infusion of KRAS-G12D-specific CD8 lesions . A solit clones. . One patA related approach involves the use of RAS-specific vaccines. One approach involves the intradermal injection of peptides along with an immune adjuvant. In a phase I/II clinical trial of 48 patients with pancreas cancer, intradermal administration of mutant RAS peptides along with a granulocyte-macrophage colony-stimulating factor (GM-CSF) adjuvant, to activate dendritic cells, elicited evidence of RAS-specific cellular immunity associated with a modest improvement in survival . In a seFor years, scientists have also tried to identify targets that are essential for the survival of cells carrying RAS mutations. Although these synthetic-lethal screens identified promising targets, results have not been translated into clinical advances . A diffeAfter years considering RAS as undruggable, the development of KRAS-G12C specific inhibitors has resulted in a renewed optimism and scientists have engaged again in the crusade of targeting RAS. The regulatory approval of KRAS-G12C inhibitors, almost forty years after the discovery of RAS, will change the treatment landscape of patients carrying this mutation. However, early clinical data and the previous experience with other targeted therapies, such as BRAF and EGFR inhibitors, indicate that the battle has not been won and that there are still many challenges ahead.It is expected that these inhibitors will have a limited efficacy as monotherapies; therefore, combination strategies will be needed. As has been discussed above, several combination strategies have been proposed and some of them are being tested in clinical trials. However, the main challenge will be to determine which combination strategies will work best for each type of tumour. Vertical pathway inhibition targeting downstream or upstream nodes of the RAS pathway could be an effective therapeutic strategy for those tumours that show KRAS dependency, whereas tumours with reduced KRAS dependency could require co-targeting of other pathways. Therefore, there is a need to identify biomarkers that can determine which patient can benefit from each strategy. Moreover, this will likely need to be done for each tumour type. Early clinical data with KRAS-G12C inhibitors show that colorectal cancer is more refractory to KRAS inhibition than lung cancer, indicating that a different approach may be needed for this type of tumour.One of the major challenges ahead is the identification of mechanisms of resistance and how they might be countered. A recent article analysing the cell-free DNA from one patient resistant to MRTX849 identified the presence of several mutations in genes of the RAS pathway, including one mutation in KRAS that affects the binding of the inhibitor . Awad anProgress during the development of KRAS-G12C inhibitors and better understanding of the cycling conformations of RAS proteins have aided the identification of inhibitors targeting other RAS alleles. Drugs targeting KRAS-G12D, one of the most prevalent mutations, are the most advanced, including Mirati’s inhibitor MRTX1133, which is progressing towards human studies. Pan-RAS-specific drugs are also being developed in order to target tumours harbouring mutations that do not have a specific inhibitor, with such compounds publicly disclosed from Revolution Medicines and Boehringer Ingelheim. However, this type of inhibitor will face the extra challenge of possible toxicity caused by blocking RAS activity in non-tumoral cells.These are exciting times for RAS research, as the development of RAS inhibitors is also helping to better understand RAS biology. The integration of knowledge obtained from both basic and clinical research is what has taken us to this point and what will allow us to, finally, remove the ‘undruggable’ label from RAS.


Patients after autologous (autoSCT) and allogeneic stem cell transplantation  are at an increased risk of COVID-19-related morbidity and mortality, compounded by an immune system weakened by the underlying malignancy and prior treatments. Allogeneic transplantation, including stem cell and solid organ transplants, requires intensive immunosuppressive prophylaxis, which may further undermine the development of a protective vaccine-induced anti-viral immunity. Herein, we report on short- and long-term antiviral immune responses in two peri-stem cell transplant recipients and a third patient who received a COVID-19 vaccination after kidney transplantation. Our data indicate that: (1) patients post-alloSCT may be able to mount an anti-COVID-19 immune response; however, a sufficient time interval between transplant and exposure may be of critical importance; (2) alloSCT recipients with preexisting anti-SARS-CoV-2 immunity are at risk for losing protective humoral immunity following transplantation, particularly if the stem-cell donor lacks antiviral immunity, e.g., vaccine-derived immunity; and (3) some post-transplant patients are completely unable to build an immune response to a COVID-19 vaccine, perhaps based on the prophylactic suppression of T cell immunity. COVID-19 infection is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and its rapid transmission around the world has resulted in a global health crisis. The SARS-CoV-2 virus contains four major structural proteins including the surface-exposed spike (S) and the internal nucleocapsid (N) proteins .An adaptive immune response occurring in the host has the potential to control the viral infection and improve patient outcomes. Our group and others have recently shown that most patients with COVID-19 indeed develop spontaneous antibody-mediated immune responses against viral proteins ,5,6 and Recently, three vaccines were authorized for the prevention of COVID-19 in the United States and over half a dozen vaccines have been authorized worldwide. These vaccines have been shown to elicit antibody- and T cell-mediated antiviral immune responses conferring protection against COVID-19 ,16,17,18A female patient in her mid-to-late 40s with a history of relapsed acute myeloid leukemia underwent a myeloablative matched unrelated allogeneic stem cell transplant 10 years prior to developing COVID-19. The patient experienced pulmonary chronic graft versus host disease (GVHD), requiring therapy for up to six years after the transplant. Of note, the patient remains in remission after the transplant and GVHD was quiescent at the time of hospital admission and in the preceding four years. The patient was intubated within two days of hospital admission for severe COVID-19. She remained on ventilator support for ~2 weeks and was treated with intravenous remdesivir. The patient was empirically treated with dexamethasone for seven days for suspected GVHD flare-up, although in retrospect she did not show evidence of GVHD. She was discharged on home oxygen after about one month in hospital. Within the first two weeks after PCR positivity, the patient developed very high IgG  and IgA The second patient, a female in her mid-to-late 70s, had a prior history of an autologous stem cell transplant 22 years earlier for relapsed follicular lymphoma. She developed a late therapy-related AML which was treated with decitabine and venetoclax. Under treatment, she developed symptomatic COVID-19 which took about two months to resolve. After she had recovered from COVID-19 and had cleared the virus, she received an alloSCT following a reduced-intensity conditioning regimen  from a h+ and CD8+ T cell counts. Unfortunately, two weeks after receiving the second dose of the vaccine, while serum antibody titers against viral control proteins  and vaccine-induced antibody levels  were comparably high, IgG and IgA titers against SARS-CoV-2 proteins S1, RBD, S2, and N were non-existent. As a result, her serum did not show evidence of any SARS-CoV-2 neutralizing activity  in 2006. She received multiple types and lines of treatments, including the proteasome inhibitor bortezomib and the immunomodulatory agent lenalidomide. In 2017, her kidney function worsened because of the underlying plasma cell dyscrasia, and she was started on dialysis. At that time, treatment with the anti-CD38 antibody daratumumab was initiated and she achieved a very good partial response. In July of 2017, she received high-dose melphalan chemotherapy followed by an autoSCT. In September of 2018, the patient received a kidney transplant and started immunosuppressive therapy with tacrolimus and mycophenolic acid. She was also intermittently treated with steroids and remained on dual immunosuppression when she received two doses of the FDA-approved mRNA-1273 COVID-19 vaccine. At that time, her LCDD was in complete remission and she had normal total IgG, IgM, and IgA serum levels and normal CD4activity .We describe here the immune responses and clinical courses of three patients with SARS-CoV-2 infection or vaccination in the context of alloSCT or solid organ transplant. The first case demonstrates that patients post alloSCT may ultimately be able to mount a strong, protective, and durable antiviral immune response; however, a sufficient time interval between transplant and exposure to the viral antigen may be of critical importance. As a consequence, clinical studies should investigate the impact of vaccination timing on the establishment of anti-SARS-CoV-2 immune responses to determine the most reasonable vaccine administration schedule for this particular patient population. Additionally, serial measurements of antibody titers will be necessary to determine the durability of viral immune responses in this severely immunocompromised group.The second case illustrates that pre-existing SARS-CoV-2 immunity may be lost following alloSCT, particularly if the stem-cell donor shows no anti-SARS-CoV-2 immunity due to the conditioning regimen, post-transplant immunosuppression use, and the establishment of the donor-derived immune system. At the same time, this case illustrates that preexisting donor immunity is effectively transferred to the recipient following alloSCT. Plausibly, this case demonstrates how donor vaccination prior to stem cell donation might confer protection from COVID-19 to patients undergoing alloSCT. Future clinical study of anti- SARS-CoV-2 immunity in recipients of immunized donors is warranted.The last case illustrates that there is a subgroup of post-transplant patients who are completely unable to build an immune response to a COVID-19 vaccine. Patients like our third case, where the solid organ transplant was performed relatively recently, are probably more likely to be affected by this unresponsive immunity. The fact that our patient had normal immunoglobulin levels and normal to high antibody titers to other microbial antigens, raises the possibility that it is not primarily a compromised humoral immune system that undermines a vaccine-induced immune protection but that the prophylactic suppression of T cell immunity  in these patients leads to a lack of T cell help for naïve COVID-19-specific B cells, resulting in an inability to mount a high-titered and durable antiviral antibody response. Furthermore, mycophenolic acid, through dual suppression of both B cell and T cell proliferation, limits both cell-mediated immune responses and antibody formation, with predictable deleterious effects on vaccine efficacy. Careful investigation of SARS-CoV-2 vaccination including a detailed and standardized assessment of immune responses, and possibly the use of additional “booster” vaccinations, in this complex group of immunocompromised patients , is warrg for 10 min and frozen immediately at −80 °C.We collected 40 mL of heparinized blood from three patients at the University of Maryland Medical Center between June of 2020 and February of 2021. Informed consent was obtained and blood samples were collected under IRB HP-00091425m, IRB HP-00057785, and IRB HP-00018133, respectively. Plasma was extracted from peripheral blood samples after centrifugation at 400× Serum antibody responses against four recombinant, full-length SARS-CoV-2 proteins expressed in human 293 cells  and viral control proteins Epstein-Barr Virus Glycoprotein gp350 (EBV), Human Cytomegalovirus Glycoprotein B (CMV), Influenza A H1N1 Nucleoprotein , Herpes Simplex Virus 1 , and tetanus toxoid  were determined by enzyme-linked immunosorbent assay (ELISA) as previously described . For theNeutralizing activity of patient sera was assessed using the cPass Neutralization Antibody Detection Kit  according to the manufacturer’s instructions. The kit detects circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the RBD of the viral spike glycoprotein with the ACE-2 cell surface receptor.


We discuss innovativeapplications of the UAA technology to challenges in bioengineeringand medicine.Since the establishmentof site-specific mutagenesis of singleamino acids to interrogate protein function in the 1970s, biochemistshave sought to tailor protein structure in the native cell environment.Fine-tuning the chemical properties of proteins is an indispensableway to address fundamental mechanistic questions. Unnatural aminoacids (UAAs) offer the possibility to expand beyond the 20 naturallyoccurring amino acids in most species and install new and useful chemicalfunctions. Here, we review the literature about advances in UAA incorporationtechnology from chemoenzymatic aminoacylation of modified tRNAs to  Expanding the mutagenesis repertoire beyond the 20naturally occurring amino acids in most species, unnatural amino acids(UAAs) enable site-specific installation of new and useful chemicalfunctions, fluorescence, ligand binding, cross-linking, or photocaging,for example. There are still challenges to UAA mutagenesis being aneveryday technique for biochemists. In this Perspective, we highlightthe 2013 work of Chatterjee and Schultz, key historical papers inthe field, and key perspective papers that illustrate future directionsbeing charted by researchers in the field. This Perspective is notmeant to stand in for comprehensive reviews published by researchersin the field.5 integratedconceptual advances in orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNAgeneration, multisite incorporation, and flexibility of codon usagein a minimalist, optimized system for incorporation of UAAs in livingcells. UAAs are incorporated into proteins in live cells by bio-orthogonalaaRS enzymes evolved to bind the UAA and its unnatural tRNA but notinteract with the naturally occurring amino acids or tRNAs or tRNAs 1. The un5 advancedthe field by streamlining multisite UAA incorporation into a simple Escherichia coli transformation with two plasmids. One plasmidpUltraII encoded one copy each of two orthogonal aaRS/suppressor tRNApairs: amber (UAG) suppressing Methanococcus jannaschii tyrosyl (MjTyr)-derived aaRS/tRNACUA and optimized ochre (UAA) suppressing Methanosarcina barkeri pyrrolysyl (MbPyl)RS/Methanosarcina mazei pyrrolysyl (MmPyl)-tRNAUUA. The secondplasmid encoded target protein green fluorescent protein (GFP) containingamber and ochre nonsense codons (GFP-3TAG-151TAA) to direct the incorporationof two unique UAAs into a single protein. This was previously intractabledue to the requirement for multiple copies of aaRS or tRNA expressioncassettes to incorporate a single UAA. The predecessor of pUltraII,pEVOL, encoded one copy of a MjTyr-derived optimizedamber suppressor tRNA and two copies of MjTyrRS toincorporate UAAs into GFP151TAG.7 With this constructin the presence of UAA p-azido-l-phenylalanine(pAzF), the suppression efficiency for a single amber codon reachedapproximately 80% of that of wild-type GFP.7 Incorporation of two UAAs, p-acetyl-l-phenylalanine(pAcF) and Nε-Boc-l-lysine(eBK), into GFP-3TAG-151TAA using pUltraII achieved 20–25%of wild-type GFP expression.5Chatterjee et al. (Schultz)l-lysine (AzK), pAcF and eBK, pAzF, and eBK,and O-methyl-l-tyrosine (OMeY) and eBK , as a substrate.11 The flexibility of the ribosomal peptidyl transferasecenter (PTC) to unusual chemistry was further shown by Fahnestockand Rich. They demonstrated that the translational machinery couldsynthesize oligomers containing multiple ester bonds using chemicallyconverted hydroxyPhe-tRNAPhe.12 However, each of these experiments was only possible because ofan idiosyncratic route to the UAA-tRNA: chemical reduction of Cys,enzymatic charging of d-Tyr, and hydroxylation of the aromaticPhe residue. What was missing was a general method for producing theUAA-tRNA.Researchers began exploring thepossibility of using modified tRNAsto incorporate UAAs soon after the discovery of the tRNA adaptor.Nε-amine of Lys-tRNALys with N-hydroxysuccinimide ester-azidobenzoic acid generated Nε-azidobenzoyl-Lys-tRNALys.13 Modified UAA-tRNA interfaced with endogenoustranslational machinery and was incorporated in place of or in competitionwith endogenous unmodified Lys in rabbit reticulocyte lysate. Becausemost target proteins contain multiple Lys residues, the modificationcould not be restricted to a single site, thus resulting in multisiteincorporation of the photoactivatable Lys UAA.13Right from the start there was significant interestin being ableto incorporate biophysical probes into proteins using UAA mutagenesis.There were foundational approaches to the task of incorporating uniqueside chains. For instance, Johnson and colleagues modified Lys afterit had been enzymatically ligated to tRNA. Acylation of the 14 T4 RNA ligase transfers an aminoacyladenylate moiety from N-blocked(with o-nitrophenylsulfenyl) aminoacylated P1,P2-bis(5′-adenosyl)diphosphatesto tRNAs lacking the 3′-terminal adenosine. However, a largemolar excess (>200-fold) of aminoacylated nucleotide derivativeswererequired for good yields, so they optimized the synthesis such thatan only 20-fold molar excess was necessary, using N-acetylaminoacyl pCpA derivatives instead.15 The modified chemical aminoacylation was used to acylate tRNAPhe with both d- and l-Phe, d- and l-Tyr, and N-acetyl-dl-β-Phe.Misacylated tRNAs can participate in peptide bond formation, consistentwith the adaptor hypothesis, but efficient dipeptide formation witha poly-Phe message occurred primarily with l-Phe, l-Tyr, and, interestingly, β-Phe, with l-PhetRNAPhe as the A-site tRNA.15In 1978, Hecht and co-workers established a generalprocedure forthe chemoenzymatic aminoacylation of tRNAs.E. coli tRNAPhe charged with a photoactivatable cross-linker UAA, l-4′-[(3-trifluoromethyl)-3H-diazirin-3-yl]phenylalanine.16 However, protein yields were often low due to the stoichiometricnature of chemically acylated tRNAs, and modifications were limitedbecause there were no general methods for UAA-tRNA synthesis. Johnsonand Brunner’s methods both allowed more flexibility in therange of biophysical probes that could be attached to either Lys orPhe; however, they still were not general methods for UAA-tRNA synthesis,and they led to uncontrolled multisite incorporation of the biophysicalprobes.16An alternative approach was demonstrated by Baldini and colleaguesin 1988. Prior to this work, due to protection of the amino groupduring pCpA ligation, the chemically misacylated tRNAs could not bindthe ribosomal A site and be incorporated into a growing polypeptidechain; thus, only dipeptides could form. By introducing a transientBoc protection/deprotection into the UAA-tRNA ligation, they demonstratedsynthesis of functional in vitro incorporation of UAAs by the Schultz lab , non-acylatedtRNACUA, and [3H]Phe]. No β-lactamaseactivity was observed, and there was no band corresponding to [3H]Phe-incorporated β-lactamase by SDS-PAGE. Significantly,they showed using [3H]Phe-tRNACUA and HPLC analysisof trypsin-digested β-lactamase that the [3H]Phewas incorporated only at position Phe66, demonstrating not only efficientincorporation of the UAA [3H]Phe in response to the UAGcodon but also that the UAA [3H]Phe was not scrambled withother natural amino acids.17Noren etal. (Schultz) showed the incorporation of three differentUAAs in the active-site residue Phe66 in β-lactamase and kineticcharacterization of these variants. They prepared an amber suppressortRNA using anticodon loop replacement of yeast tRNAl-3-iodo-tyrosine into a 16-residuepolypeptide.20 They prepared a semisynthetic,non-hypermodified E. coli glycyl tRNACUA nonsense suppressor tRNA acylated with l-3-[125I]tyrosine and incubated with the message containing UAG at position9 in rabbit reticulocyte lysate. The translation product was purifiedand sequenced to unambiguously determine the site specificity of incorporation.Nonsense suppression was due entirely to the added synthetic suppressorbecause they could not detect read-through by endogenous aminoacyl-tRNAs(aa-tRNA).21Bainand co-workers used a strategy similar to that of the Schultzgroup to incorporate 22 Unprotected pdCpA was selectively aminoacylatedin high yield with the cyanomethyl ester (CME) of N-blocked amino acid and ligated to tRNA. The photolabile nitroveratrylprotecting groups for the α-amine and side chain functionalgroups enabled the aa-tRNA to be deprotected photochemically. Thisreaction produces high yields of stable, unblocked aa-tRNA that canbe used directly in a purified translation system. The approach greatlysimplified the synthesis of UAA-pdCpA to one high-yield (76–87%)step.22A key issuecontinued to be the lack of a general method for synthesizing theUAA-pdCpA 4. A key N-biotinyl-l-methionyl group from the 3′-endof a donor hexanucleotide, 5′-pCAACCA-3′, to the 5′-hydroxylgroup of the ribozyme.24 Suga, Szostak, and co-workersgenerated aaRS-like ribozymes with two catalytic domains: one thatrecognizes the amino acid substrate and self-aminoacylates its 5′-hydroxyland the other that binds the tRNA and transfers the aminoacyl groupto the 3′-end. This ribozyme acts as a synthetase that cancharge tRNAfMet with Gln or Phe.25 CME was chosen as a leaving group on the amino acid because it hasno hydrogen bond donors or acceptors that could interact with theribozyme. Active RNAs could be isolated from the pool by selectionwith N-biotinyl-l-glutaminyl-CME and subsequentpull-down with streptavidin.25An alternative approachto tRNA aminoacylation was catalytic RNA,or ribozymes. Natural ribozymes catalyze trans-esterification reactionsof phosphodiester bonds. Szostak and co-workers isolated catalyticRNAs with acyl transferase activity, like that of the PTC, from poolsof random RNA sequences. They selected for enhanced transfer of an de novo catalystfor tRNA acylation using aaRS-like RNA molecules called Flexizymes(Fx) and mutants thereof.26 They noticedthat Fx recognizes neither the leaving nor the amino group of thesubstrate, but rather the aromatic functionality of the amino acidside chain and the carbonyl group of the ester. To improve binding,they redesigned substrates incorporating an aromatic ring in the leavinggroup. They used dinitrobenzyl ester (DBE) and the more activatedchlorobenzyl thioester (CBT). Because DBEs are less hydrolyticallylabile than CBTs, DBEs were used in further experiments. Enhancedinteraction between Fx and the substrate significantly enhanced tRNAacylation efficiency and enabled incorporation of citrulline, Nε-acetyl-l-lysine, Nε-biotinyl-l-lysine, p-iodo-l-phenylalanine, (S)-3-isopropyllacticacid, and (S)-3-phenyllactic acid into short (nine-aminoacid) FLAG-tagged peptides expressed in an E. coli cell-free translation system, Protein synthesis Using RecombinantElements (PURE),27 by amber-programmedframeshift suppression and sense codon reassignment .26 This breakthrough represented apowerful tool for enhancing the range of UAAs that could be ligatedto tRNAs and incorporated into polypeptides in vitro.TheSuga lab generalized the ribozyme Another focus of the field followingNoren et al. (Schultz) in 1989 was pushing the boundaries of whatUAA structures and functions could be incorporated and using theseUAAs to address important protein structure and function questions.Key questions of enzyme mechanism and protein stability could be addressedby incorporating unnatural isoelectronic or isosteric analogues ofnatural amino acids at sites of interest and measuring changes inenzyme kinetics and/or protein denaturation.28 Specifically, they tested if different UAAs could be incorporatedat position Ala82 in T4 lysozyme (T4L) and how the UAAs affected thestability of T4L. Ala82 is a surface residue located between two helices,distant from the active site. The structure and electronics of theUAA significantly affected its use as a substrate by the translationalmachinery. The incorporation efficiencies were as follows: none detected(ND) for d-Ala and 30% (suppression efficiency) for lacticacid; N-alkyl amino acids, <5% for azetidine 2-carboxylicacid, 43% for pipecolic acid, 24% for N-methyl-alanine,and <5% for N-ethyl-alanine; α,α-disubstitutedamino acids, 28% for cyclopropylglycine and 23% for α-aminoisobutyricacid. Interestingly, changing the amino acid structure and electronicschanged the apparent yield of protein synthesis; because an E. coli S30 crude cell extract was used, the mechanism ofthis decrease in yield could not be determined at the time. The stabilitiesof the resulting UAA-substituted T4L proteins were determined by thermaldenaturation as measured by circular dichroism.28 These UAA backbone analogues largely changed the stabilityof T4L as would be predicted.Building on earlierwork with modified nucleosides and tRNAs, Ellman and Mendel workingwith Schultz explored the tolerance of the translational machineryto changes in the amino acid structure.29 Staphylococcalnuclease (SNase) accelerates the hydrolysis of phosphodiester bondsin nucleic acids approximately 1016-fold. One hypothesiswas that general base catalysis underpins this enormous rate accelerationwhere Glu43 in SNase acts as a general base to activate a water moleculefor attack on the phosphodiester backbone of DNA. However, when Glu43was replaced with isoelectronic and isosteric analogues, Arg, S-4-nitro-2-aminobutyric acid, S-2-amino-5-hydroxypentanoicacid, aminoethylhomocysteine, and citrulline, differing only by beingpoorer bases, the kinetics of SNase were virtually unchanged relativeto those of the wild-type. A significant accomplishment at the timeworking with E. coli S30 cell extracts, a structureof the enzyme substituted with homoglutamic acid at site 43 was obtained.Combined, the kinetic and structural data suggested that Glu43 mayinstead play a structural role, fixing the conformation of a nearbyloop.29Judice and Schultz used UAA incorporationto make more precisechanges in amino acid structure than possible with the natural aminoacids to probe enzyme mechanism.30 Thus,Cornish, Hahn, and Schultz incorporated a small ketone handle thatcould subsequently be modified to form an oxime or other unnaturallinkage to the biophysical probe in what has come to be called bio-orthogonallabeling.31 They incorporated keto aminoacids 1 (5% suppression efficiency) and 2 (30% suppression efficiency) in sites Ser44 and Ala82 in T4L, twosites known generally to give high suppression efficiencies , ochre (TAA), and opal (TGA). Alternate codonswould increase the number of UAAs that could be incorporated in asingle protein. In the 1990s, frameshift codon suppression was exploredas an alternative to nonsense suppression 6. When tACCU and Ala-tRNACCUA incorporatedAla into E. coli dihydrofolate reductase (DHFR),shown by a restoration of enzymeactivity to 40% and 15% of wild-type activity, respectively.32 Repurposing of rare codons was another strategyfor expanding options for UAA incorporation. AGG was used as an alternativecodon for incorporation of the photoactive UAAs p-phenylazophenylalanine, 2-anthrylalanine, 1-naphthylalanine, 2-naphthylalanine,and p-biphenylalanine into a polypeptide expressedin an E. coli S30 extract, where AGG is rare (<3%).33 The Sisido lab extended this work to incorporatethe UAAs nitrophenylalanine, 2-naphthylalanine, p-phenylazophenylalanine, and 2-anthrylalanine into streptavidin (Tyr83→ AGGU) through frameshift suppression in E. coli S30 extracts.34 CGGG was found to workmore efficiently than ACCU in further studies.36Using this strategy, frameshiftsuppressor Ala-tRNAE. coli S30 cell extract with nitrophenylalanineefficiently incorporated into streptavidin using the codons AGGU,CGGU, CCCU, CUCU, CUAU, and GGGU.37 Furtherutility of this approach was demonstrated by the incorporation oftwo UAAs, nitrophenylalanine and 2-naphthylalanine, into streptavidinusing the quadruplet codons CGGG and GGGU.37 Incorporation of two fluorescent UAAs in E. coli DHFR was achieved by a combination of quadruplet codon and ambersuppression.38 Specifically, the fluorescentUAA 7-azatryptophan was incorporated with a CGGG quadruplet decodingtRNA and acceptor Nβ-dabcyl-1,2-diaminopropionicacid by amber suppression in E. coli DHFR.38More options for different quadruplet codons were demonstratedin an 39Sense codon reassignment, or genetic codereprogramming, has beenexplored as an alternative to nonsense suppression. Nonsense codonsuppression is limited to two UAAs because there are only three nonsensecodons and one must be used for translation termination. It wouldbe of enormous practical utility and would allow fundamental questionsabout the genetic code to be addressed if sense codons could be reassigned.Forster, Tan, Cornish, and Blacklow established the concept of geneticcode reprogramming of multiple, adjacent sense codons by reassigningthree sense codons to UAAs using chemoenzymatically charged tRNAsin a reconstituted translation system lacking aaRSs.E. coli S30 in vitro extract with competing aa-tRNAs and aaRSs thatcould hydrolyze noncognateaa-tRNA pairs and recharge the tRNA with the cognate amino acid, wemade a purified in vitro translation system ourselvesbased on published protocols in the ribosome mechanism field. TheUAA-tRNAs were prepared using the chemoenzymatic methods being usedby the Schultz group at the time. Because it was the first attemptto modify multiple sense codons, we started with three conservativeside chain modifications: O-methyl-l-serine,2-amino-4-pentenoic acid , and 2-amino-4-pentynoic acid(propargyl glycine). Tracking the peptide synthesis using [35S]Met-tRNA, [3H]Glu-tRNA, and authentic peptide markersprepared by solid-phase peptide synthesis, translation of a peptidewith five of the same UAAs in a row was demonstrated. Finally, translationof a peptide with three different UAAs in a row each in response toa different sense codon was achieved. Together, this work showed forthe first time that multiple sense codons could be reassigned allowingfor translation of unnatural oligomers, a direction we argue belowwill become increasingly important to the field.Rather than using an 40 The Suga lab establishedthe mRNA-encoded incorporation of multiple, consecutive amino acidanalogues for the in vitro synthesis of unnaturalpolypeptides using a combination of frameshift suppression and sensecodon reassignment  in the Fx/PURE reconstitutedtranslation system with depleted aaRSs and cognate amino acids.26 The ability to control installation of multipleUAAs significantly increased the diversity of peptides that can besynthesized and screened for therapeutic properties. Suga has goneon to do a lot of work in this area, for example, incorporating multiple,consecutive amino acid backbone analogues into the peptide backbone,including α-hydroxy amino acids,41Nα-methylated amino acids,42 and, more recently, d-amino acids43 and β-amino acids.44 However, to date, sense codon reassignment is limited to in vitro translation systems.Around thesame time, Josephson, Hartman, and Szostak establishedribosomal synthesis of nonribosomal peptide-like molecules containing10 UAA side chain analogues by sense codon reassignment, significantlyincreasing the number of UAAs that can be incorporated into a singlepolypeptide.E. coli S30,wheat germ, or rabbit reticulocyte extracts. Methods developed inthe laboratories of Dougherty and Lester pioneered the use of UAAsin Xenopus oocytes, where they injected UAA-pdCpA-ligatedtRNA and mRNA encoding the protein of interest with an amber codon,with a particular focus on eukaryotic ion channels.45 Specifically, Lummis and co-workers explored the role ofa highly conserved Pro at site 8 (Pro8) in cation-selective 5-hydroxytryptaminetype 3 receptors. Pro8 acts as a hinge in the loop between the secondand third transmembrane helices, a region that interacts with theextracellular ligand binding domain and was hypothesized to play animportant role linking neurotransmitter binding to channel gatingthrough cis–trans isomerization of the proteinbackbone. Incorporation of Pro analogues favoring the cis conformer produced functional channels, while those favoring the trans conformer produced nonfunctional channels. Importantly,the cis–trans energy gap of the Pro analoguewas strongly correlated with channel activation, suggesting cis–trans isomerization of this single Pro acts asa gating switch between open and closed channel states.45 These experiments built upon their earlier workoptimizing the pdCpA ligation chemistry for backbone analogues, suchas α-hydroxy amino acids.46To date, UAAs were incorporated into Xenopus, Rodriguez and co-workersdemonstrated multisite incorporation of UAAs into nicotinic acetylcholinereceptors by combining nonsense and frameshift codon suppression.47 A limitation of chemoenzymatically charged tRNAsis that they cannot be reacylated once inside the cell, capping theamount of protein that can be generated. An ideal system would includeall of the necessary components genetically encoded in the cell. Amain challenge of eukaryotic genetic code expansion is the fact thattranslational machinery is not well conserved between prokaryotesand eukaryotes.With respect to expanding the repertoire of codons for incorporationof multiple UAAs in E. coli through directed evolution of an orthogonalaaRS/tRNA pair.48 This advance addressedmultiple technical challenges in the field. One, it addressed thetechnical difficulty of preparing the aminoacyl-tRNA chemoenzymatically.Two, it removed the limit on protein yield imposed by use of an in vitro translation extract. Significantly, the UAA technologycould now be adopted by a non-expert in the technology.The next big breakthrough in the field was fullgenetic encodingof UAA incorporation components in live cells. In 2001, Wang et al.(Schultz) reported a general method for incorporating UAAs into proteinsin 49 The naturally orthogonal MjtRNACUATyr and MjTyrRS were used as the starting point. First, they useda negative selection based on suppression of a UAG codon in the toxicRNase Barnase in the absence of MjTyrRS to selectfor MjtRNACUATyr variants that could not be aminoacylatedby the endogenous natural aaRS enzymes.49 Next, the winners were subject to a positive selection for MjtRNACUATyr variants that could suppress amber mutations in β-lactamasein the presence of MjTyrRS.49 Finally, to generate the orthogonal MjTyrRS, E. coli cells were transformed with a library of MjTyrRS genes and subjected to a positive selection forsuppression of a UAG codon in chloramphenicol acetyltransferase (CAT).The library was subject to both positive and negative selection  to yield pairsthat incorporate the UAA but not natural amino acids in response tothe UAG codon. They used the resulting orthogonal aaRS/tRNA pair toincorporate O-methyl-l-tyrosine into DHFRexpressed in E. coli.48Thekey conceptual advance was the positive and negative selectionstrategy for evolving the orthogonal tRNA and aaRS.in vitro cell extracts and broadly enabledengineering UAA biosynthetic machinery in live cells.48 There were some hurdles in getting there, with differentselection approaches being less successful.50 Once a general method for generating mutant aaRS-tRNA pairs wasestablished, issues that needed to be addressed were how to generatenew orthogonal aaRS-tRNA pairs for UAA incorporation in model systemswhere large mutant libraries cannot be made, such as mammalian cells,and the lack of suitable starter aaRS–tRNA pairs orthogonalin these systems. The transfer of E. coli TyrRS-tRNACUA and LeuRS-tRNACUA to yeast and mammalian cellswas feasible; however, tRNA expression was a major hurdle at the time.The Wang lab developed a general method for expressing orthogonaltRNAs in mammalian cells using type 3 Pol III promoters, and thisis the general method currently being used in the field for UAA incorporationin yeast, mammalian cells, and various animals.51 Another challenge was that the size of the aaRS bindingpocket limited the stereochemical diversity of UAAs that could beincorporated. A breakthrough in the field was made by the Wang labwith the finding that mutation of the Methanosarcina PylRS binding pocket can generate more flexible substrate specificityfor Phe and Tyr analogues with bulky conjugated rings or long azobenzeneside chains.52While still limited by the need to purify the UAA-incorporatedprotein out of the cell, this work eliminated the problem of low proteinyields with Saccharomyces cerevisiae.53 They exploited the fact that E. coli tyrosyl tRNACUA can be expressed in yeast and is a poor substrate for the S. cerevisiae aaRSs. They evolved TyrRS in yeast for incorporationof acetyl, benzoyl, azido, and iodo-Phe analogues, as well as O-methyl-l-tyrosine, into human superoxide dismutase.53 This system became the basis for directed evolutionof aaRSs for UAAs that could be readily transferred to mammalian cells.Genetic encoding of orthogonal synthetase–tRNA pairs in mammaliancells and animals, including Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus, followed suit.60The Schultz lab demonstratedthe first fully genetically encodedUAA incorporation system in eukaryotes when they incorporated UAAsin 61 Azidohomoalanine (AHA) is an azide-bearingmethionine analogue that can be incorporated at methionine codonsand tagged with an alkyne-affinity tag using copper-catalyzed azide-alkyne[3+2] cycloaddition for the identification of AHA-labeled proteinsvia mass spectrometry.61 Another methioninesurrogate, alkyne-bearing homopropargylglycine (HPG), was used intandem with AHA to fluorescently label newly synthesized proteinsin rat hippocampal neurons by strain-promoted cycloaddition.62 Pulse-chase experiments enabled fluorescentlabeling of two distinct proteomes synthesized sequentially in timesuch that the dynamics of protein synthesis and fate could be monitoredin neurons.62 Thus, they could addressimportant questions about the role of newly synthesized proteins inneuron function without the need for selective incorporation of theUAA.Asearly as 2006, Tirrell and Schuman established metabolic labelingwith click handle UAAs as a nonspecific, heterogeneous multisite UAAincorporation method for tagging newly synthesized proteins in mammaliancells.51 UAA esterification to increaseUAA bioavailability,63 and optimizing tRNA/synthetaseaffinity to increase the level of UAA incorporation.64 Encouraging progress was achieved in this research areathrough the combined use of these optimized methods.4 In their letter to the editor of Cell Research, Ye, Wang, Li and co-workers reported the introduction, maintenance,and transmission of the genetic material for code expansion in mice.In this work, they integrated the orthogonal pAzFRS/tRNACUA pair into the mouse genome.59 They demonstratedthat, in the presence of pAzF, the suppressor tRNA can decode theUAG amber codon to express a dual fluorescent reporter eGFP-TAG-mCherryin neurons and bone marrow cells of mice.59Efficient incorporation of UAAs into proteins in animalshas also been a critical challenge. To improve the efficiency, manydifferent methods have been researched, including using the type 3polymerase III promoter to more efficiently express orthogonal prokaryotictRNAs,l-polypeptides. The logic of making backbone analogue UAA incorporationwork in vivo began with the discovery or generationof ribosomes that can accommodate these UAAs followed by testing ofknown aaRS/tRNA pairs for charging them. In 2016, Schepartz and Söllincorporated β3-amino acids into full-length DHFRin E. coli.65 Previously,Dedkova and Hecht had found that ribosomes from some erythromycin-resistant E. coli mutants could tolerate the incorporation of β3-amino acids into full-length DHFR in vitro.66 Building on this work, Schepartz andSöll highlighted the unexpected flexibility of the endogenous E. coli translational machinery to β3-aminoacid backbone analogues when they demonstrated incorporation of β3-amino acids into DHFR expressed in E. coli harboring a plasmid encoding mutant ribosomes from erythromycin-resistantstrains.65 Significantly, they demonstratedthat β3-(p-Br)Phe and β3-Gly could be charged by endogenous aaRS enzymes, with PheRSbeing the most tolerant of these substrates. Furthermore, wild-typeEF-Tu interacted efficiently with β3-Phe-tRNAPhe for delivery to the ribosome. To improve the efficiencyand selectivity for β3-amino acid incorporation,a library of peptidyl transferase center 23S rRNA mutant ribosomeswere screened for erythromycin resistance and β-puromycin sensitivity,resulting in a new mutant P7A7 that imparted 3-fold higher levelsof β3-amino acid incorporation over those of thepreviously discovered mutants.65 Trypticdigest of DHFR peptide fragments containing either α-Phe orβ3-(p-Br)Phe at F128 showed a 10-foldlower level of incorporation of the β3-amino acidversus α-Phe.65One of the most exciting classes of UAAscontinues to be backbone analogues because of the potential to extendthe power of genetic encoding to oligomers other than α-in vivo, it has been difficult for scientists to producepeptides containing d- and β-amino acids by UAA incorporation in vitro. By tuning tRNA sequence and concentrations ofnative initiation (IF2) and elongation factors (EF-Tu/Tsand EF-G), in 2017, the Suga lab increased the yield of a d-Ala-d-Ala-containing peptide by >5-fold and incorporated10 consecutive d-Ser residues into a peptide chain.43 The existence of mutant E. coli ribosomes that enhance d-amino acid incorporation in vitro indicates potential for in vivo incorporation of d-amino acids.67 Similarly, in 2018, the Murakami lab improved incorporation of multipleβ-amino acids, producing peptides with natural amino acid spacersbetween two or three β-amino acids in their optimized translationsystem.68 Translating the in vitro advances in incorporation of backbone analogue UAAs to cells shouldcatch on as more suitable aaRS/tRNA pairs are established. In 2019,Dedkova and Hecht found that wild-type PylRS could incorporate a fluorescentoxazole UAA lacking an asymmetric center or α-amino group. MreB(Leu13TAG) was co-expressed with PylRS in an E. coli strain with modified ribosomes that could incorporate dipeptidesand dipeptidomimetics.69 It is becoming clearer how malleablethe translational machinery is for incorporation of more exotic UAAs.Importantly, this research signals the possibility of finding moremutant ribosomes and aaRS/tRNA pairs for backbone analogue UAA incorporation in vivo. One direction the field is going is to combinean expanded pool of UAA backbone analogues with an expanded pool oforthogonal codons and engineered orthogonal ribosomes for geneticencoding of unnatural oligomers in vivo.Underscoringthe importance of recent work incorporating backbone analogue UAAs O-phosphoserine (Sep) is the most abundant phosphorylatedamino acid in eukaryotes. It is synthesized post-translationally byacylation of tRNACys with Sep by SepRS, an aaRS uniqueto methanogenic archaebacteria. Park et al.  made an amber suppressing tRNASep and, critically,evolved an EF-Tu mutant, EF-Sep, that could bind Sep-tRNASep for site-specific incorporation of Sep into proteins in E. coli.70 SepRS, tRNASep, and EF-Sep together allow E. coli to read UAGas a Sep codon; they synthesized mitogen-activated ERK activatingkinase 1 (MEK1) with Sep incorporated at a key modified residue Ser218by amber suppression in vivo.70 In addition, it would need to be established that Sep wasincorporated at only the intended UAG residue. Similarly, tyrosinephosphorylation is a critical PTM in cellular signal transduction.In 2017, Luo et al. (Schultz and Wang) incorporated O-phosphotyrosine (pTyr) and its nonhydrolyzable analogue, 4-phosphomethylphenylalanine,into recombinant proteins by amber suppression in E. coli.71 Around the same time, Hoppmann andco-workers  incorporated a neutral pTyranalogue into recombinant proteins in E. coli byamber suppression; deprotection results in a native, negatively chargedpTyr at the desired site.72 Multisite incorporationof these UAAs would enable modeling of multiple phosphorylated residuesof a protein.72Anotherclass of UAA analogues that could be highly impactful isepigenetic protein modifications. Research on the functional effectsof specific epigenetic protein modifications is hindered by the difficultyof synthesizing post-translationally modified target proteins in cells. in vivo. Wild-typeexpression levels of UAA-modified protein cannot be realized becausesuppressor tRNAs compete with release factors (RFs) for the stop codon.Church, Isaacs, and co-workers have pioneered the breakthrough inthis area by developing genomically recoded organisms (GROs). Uponrecoding of the entire genome of E. coli such thatall UAG stop codons are mutated to the UAA stop codon and deletionof RF1, the “blank” UAG codon could then be reintroducedas a sense codon for highly efficient incorporation of a UAA.73 The new genome enabled this new strain of E. coli C321.ΔA to exhibit increased resistance toviral infection by blocking the translation of viral proteins.73 The Church lab has led this area of researchtoward creating GROs with expanded capabilities. Ostrov and co-workers(Church) created E. coli with a 57-codon genome inwhich all 62214 instances of seven codons were replaced with synonymouscodons in all protein-encoding genes.74 When the recoded codons’ respective tRNAs and release factorare removed, up to four orthogonal UAAs could be introduced into theorganism.74 The increased yield and specificityof UAA incorporation in GROs should empower efforts for industrialUAA-modified protein production and more representative invivo experiments with UAA-modified proteins.Low yield is a significanthurdle to studying proteins modifiedwith UAAs by amber suppression E. coli strain C321.ΔAhas been used subsequently to advance techniques for studying post-translationalmodifications with UAAs. Isaacs and Rinehart conducted a proteome-wideinvestigation of the role of phosphorylation of human proteins in vivo.75 They genetically encodedSep70 in a synthetic human phosphopeptidelibrary expressed in C321.ΔA and identified proteome-wide phosphorylation-dependentinteractions using bimolecular fluorescence complementation in cells.75 In contrast, for in vitro studiesof the regulation by phosphorylation of 26S proteasome subunit RpnI,Sep was incorporated into RpnI(361TAG) expressed in E. coli strain C321.ΔA, allowing for purification of homogeneouslyphosphorylated RpnI.76 To transfer thistechnology to mammalian cells, Chin and co-workers demonstrated orthogonalityin mammalian cells of an evolved SepRS/tRNACUA pair77 based on the original by Park and co-workers.78 The Sep incorporation system in mammalian cells was completed byco-expression with eRF1(E55D) (a eukaryotic RF more permissive ofUAG read-through), creation of a eukaryotic elongation factor variantEF-1α-Sep containing mutations analogous to those of the prokaryoticEF-Sep, and deletion of phosphoserine phosphatase to increase theintracellular Sep concentration.78 If theUAA technology can be used to selectively introduce Sep (and ultimatelyother epigenetic modifications simultaneously) at multiple, definedpositions in a single protein in living cells, this will be a verypowerful tool for biologists.The genomicallyrecoded 80 and we have highlighted advances in this field and the related chemicaltagging field previously.81 Here we focuson the significant challenge of efficient and robust fluorescent labelingof intracellular proteins in mammalian cells, highlighting the systematiccomparison of fluorescent UAA labeling by bio-orthogonal click chemistrytechnologies from Peng and Hang.82 Whilethere has been success in labeling intracellular proteins in livemammalian cells using bio-orthogonal strain-promoted click chemistry,84 the majority of published work still focuses on cell surface proteins.Peng and Hang wanted to observe the localization and trafficking ofthe small membrane-associated protein interferon-inducible transmembraneprotein 3 (IFITM3) in cells. Given that GFP is twice the size of IFITM3,they took advantage of the small size and modularity of organic fluorophoresafforded by the UAA technology. Site-specific incorporation of thecommercially available trans-cyclooct-2-ene lysine(2′-aTCOK) into IFITM3 by amber suppression with the MmPylRSAF/tRNA pair87 led to themost efficient and specific labeling with monosubstituted tetrazine(Tz) fluorophores, e.g., tetrazine silicon rhodamine (H-Tz-SiR), inlive mammalian cells  is anindispensable technique for localization and mechanistic studies ofprotein targets inside cells. However, the large size of FPs (27 kDa)limits tagging to the protein termini and the targets that can bestudied. The most efficient chemical tags also require protein tags,and the peptide chemical tags rely on two-step labeling with bio-orthogonalchemistry. The holy grail of fluorescent labeling would be directand selective incorporation of a fluorescent UAA with a high photonoutput in mammalian cells. The challenges are having the ribosomeaccept a large fluorophore as a substrate, minimal nonspecific labelingof the fluorescent amino acid in the cell or incorporation into otherUAG codons, or rapid and selective bio-orthogonal chemistry; one indicationthat the labeling technology meets these criteria is that it can beused to image a typical cellular protein at ∼1 μM concentrationsfreely diffusing intracellularly with single-molecule resolution.Comprehensive reviews of this literature have been published,an cells 7.82 H-Tz88 The small size, the flexibility of the incorporationsite, and the keen environmental sensitivity of Aladan provide unparalleledspatial resolution for detecting the electrostatic properties of variousregions of this protein.88The alternative to bio-orthogonal click chemistrylabeling is directincorporation of a fluorescent UAA. The benefit of this approach isease. All that is required is transfection of the aaRS/tRNA and targetconstructs, incubation with the UAA, and washing out excess UAA. Thedrawbacks are the limitations on the size of the fluorescent sidechain due to the aaRS binding pocket and, thus, the tendency for thesefluorophores to be relatively blue-shifted and dim. Nonetheless, site-specificfluorescent labeling of proteins with fluorescent UAAs is built onthe foundation of direct incorporation of a single fluorescent aminoacid. Jan and Cohen established the state of the art with their incorporationof environmentally sensitive fluorescent UAA Aladan into the B1 domainof streptococcal protein G (GB1) by solid-phase synthesis.In vivo incorporation of Aladan analogues, suchas Anap, was enabled by the establishment of aaRS/tRNA pairs for thisUAA, derived from E. coli LeuRS/tRNACUA.89 Anap undergoes a blue-shift in fluorescenceemission in increasingly hydrophobic environments, making it promisingas a sensor of protein conformational change. Shandell, Cornish, andKass demonstrated the feasibility of sensing the conformational changeof a population of UAA-modified cardiac voltage-gated sodium channelsexpressed in live mammalian cells through incorporation of Anap intothe inactivation gate, a dynamic ∼50-amino acid intracellularlinker.90 Since the foundational work ofDougherty and Lester, ion channel physiologists have embraced theUAA technology in oocytes.92 Significantly, Puljungand co-workers incorporated Anap into KATP channels inlive mammalian cells, enabling voltage-clamp fluorimetry experimentsin this new, possibly more physiologically relevant context.93 Ligand binding or conformational change measuredby Anap environmental sensitivity can now be coupled to functionalchanges in channel gating measured by electrophysiology in mammaliancells.93 More impactful applications ofthe technology are being published as technical challenges are overcomeand efficiency and ease of use improved. When multiple fluorophoreswith single-molecule resolution can be incorporated selectively intomammalian cells, the UAA technology will be a powerful tool for studyingbiological mechanism in living cells.9495 To model biological conditions that could explainthe transition from an RNA world, Schultz and co-workers engineeredchimeric E. coli in which 40% ribonucleotide versusdeoxyribonucleotide could be incorporated into the genome when thesize of the pool of deoxyribonucleotide triphosphates in the cellwas significantly decreased in concert with defects in DNA repair.96 In a similar fashion, Schultz and co-workersmodeled the central hypothesis of endosymbiotic theory that mitochondriacould have evolved from prokaryotes entering host cells and beingmaintained as endosymbionts. They engineered chimeras of E.coli and S. cerevisiae in which mutant E. coli live in the cytosol of and provide ATP to a respiration-deficientyeast mutant or yeast provide thiamin to a resident E. coli thiamin auxotroph.97 SSOs can be generatedby codons containing unnatural base pairs or through the engineeringof sense codon usage. Zhang et al. described an optimized SSO thatstores genetic information using DNA containing two additional letters,which form a third, unnatural base pair, dNaM (mRNA codon) and dTPT3(tRNA anticodon).98 This expanded geneticcode enables decoding of new codons to direct site-specific incorporationof UAAs into proteins in E. coli.98 Recently, the Chin lab announced the creation of a SSOwith a 61-codon genome. Creation of such a synthetic, minimally recoded E. coli genome by compressing synonymous codons addressesorigins of life questions and biological mechanism and is enormouslyuseful for therapeutic applications.99Wonderfullycreative, the UAA technology will inspire myriad newdirections. One particularly expansive direction is to engineer notonly the amino acids but also the nucleic acids, organelles, and otherparts of the cell to give rise to unnatural, chimeric, and semisyntheticorganisms (SSOs). In some ways, this is the counterpart to buildingan artificial cell ground up from artificial RNA parts.d- and β-amino acids.It turned out that the translational machinery may be more tolerantto unnatural substrates than previously thought. Mechanistic insightinto how these analogues interfere with translational machinery wasneeded. Leyh, Gonzalez, Cornish, and co-workers further clarifiedthe mechanism by which d-amino acids disrupt translationin a purified translation system, finding that while d-aa-tRNAcan be accepted at the A site, act as a peptidyl-transfer acceptor,and translocate the peptidyl-d-aa-tRNA into the P site, thisprocess occurs slowly.100 Furthermore,the peptidyl-d-aa-tRNA at the P site partitions ribosomesinto arrested and non-arrested subpopulations. Chemical protectionand molecular dynamics simulations demonstrated that P-site-boundpeptidyl-d-aa-tRNA traps the PTC in a conformation that isnot conducive to peptidyl transfer, providing insight into how theribosome discriminates between l- and d-amino acids.100 This mechanism of discrimination against d-amino acids appears to be similar to other peptide stallingmechanisms of the ribosome and may suggest the mechanism by whichit discriminates against UAAs generally. Further mechanistic workwith UAAs no doubt would lead to additional mechanistic surprisesand could significantly inform efforts to improve the efficiency andbreadth of the technology.Somewhatsurprisingly, there has been little integration of the incredibleadvances in our understanding of the structure and mechanism of thetranslational machinery and the UAA technology since the breakthroughpublication by Noren et al. (Schultz) in 1989. It has often been thoughtthat natural limitations of the translational machinery underlie thedifficulty in incorporation of in vivo using alternate codons. Yields of UAA-incorporatedproteins expressed in mammalian cells are significantly lower thanthose expressed in E. coli. Thus, it is an uphillbattle to yield multiply UAA incorporated proteins in mammalian cells.In 2013, Chatterjee et al. (Schultz) developed a baculovirus-baseddelivery system for efficient incorporation of UAAs into proteinsin mammalian cells.101 Later that year,the same mammalian suppression system was applied to incorporate twodistinct UAAs (eBK and OMeY) into EGFP in HEK293T cells using TAGand TAA suppression with EcTyrRS/tRNACUA and MbPylRS/MmtRNAUUA pairs, respectively. They also demonstrated the application of dualsuppression to fluorescent labeling of antibody–drug conjugates(anti-Her2-IgG-nAF) purified from HEK293F cells.102 In mammalian cells, nonsense suppression suffers from alow level of expression of suppressor tRNAs and competition with endogenousrelease factors seeking to truncate target protein. In 2011, Johnsonand co-workers established that knockout of release factor 1 in E. coli enables incorporation of UAAs at multiple TAG sitesin the same gene.103 In mammalian cells,by optimizing the PylRS/tRNACUA expression system and engineeringeukaryotic release factor 1, Schmied and co-workers were able to increasethe yield of protein containing UAAs at three sites by 2–4-fold.104 Multisite incorporation at unique codons selectively in vivo remains a challenge for the field.Whether the objective is to incorporatedifferent epigeneticmodifications or to synthesize an unnatural backbone oligomer, itwill be critical to be able to incorporate multiple different UAAs E. coli expressionsystems due to the lack of several blank codons and mutually orthogonalaaRS/tRNA pairs. In a breakout publication, Rackham and Chin addressedthis challenge by engineering an orthogonal ribosome in E.coli via an engineered duplicate, orthogonal Shine-DalgarnomRNA sequence/16S small ribosomal subunit RNA pair.105 Building on this work, Chin and co-workers have evolvedan orthogonal ribosome for quadruplet and amber decoding.106 Orthogonal ribosomes can be devoted to efficientdecoding of alternative codons because they are directly targetedto a corresponding orthogonal mRNA and thus do not synthesize theproteome. Through the combined use of mutually orthogonal aaRS/tRNApairs, the evolved ribosome, and corresponding orthogonal mRNA, twoUAAs were incorporated into single recombinant fusion proteins in E. coli. The cross-linking UAA p-benzoylphenylalanine(Bpa) and click handle pAzF were incorporated into glutathione S-transferase (GST)-maltose binding protein in responseto a quadruplet and amber codon. The alkyne N6-[(2-propynyloxy)carbonyl]lysine(CAK) and pAzF were incorporated into GST-calmodulin in response toa quadruplet and amber codon.106 Incorporationof pAzF and CAK into calmodulin enabled formation of a triazole cross-linkby copper-catalyzed click chemistry, demonstrating precise geneticcontrol of protein conformation with the UAA technology.One strategytoward multisite UAA incorporation at unique codonsis to take advantage of our growing structural and mechanistic understandingof the translational machinery to engineer the ribosome itself. Whenthe toolbox is expanded to include both quadruplet and amber codons,the ability of the natural ribosome to decode such codons limits theefficiency of UAA incorporation and the resulting yield of UAA-incorporatedprotein. It has been an outstanding challenge incorporating multipleUAAs in a single protein even in 107 However, the continuous exchange of the subunitsof the ribosome still limits the establishment of complete orthogonalitywith native translation (108 They demonstrated that the engineeredribosome with inseparable tethered subunits (Ribo-T) is capable ofsupporting the growth of E. coli cells, wholly orthogonal,and does not interfere with wild-type ribosomes.109 They demonstrated the unique utility of Ribo-T in studying dominantlethal mutations of rRNA, a nearly impossible task in other cell models.108 The Chin lab used a similar approach to covalentlylink the small and large ribosomal subunits by RNA linkers.111 The compatibility of the tethered ribosomal complexes with the multisiteincorporation of UAAs was evaluated by Jewett and Mankin through thefluorescence analysis of a super folder GFP (sfGFP) variant containingfive TAG codons, finding that the tethered translation system is effectivein incorporating five pAzF click handles into sfGFP.112 What remains is to show incorporation of multiple differentUAAs in a row, each in response to a different codon.Earlystrategies for achieving orthogonality involved the development oforthogonal aaRS/tRNA pairs, building of orthogonal genetic codes,and creation of orthogonal ribosome–mRNA pairs by engineeringthe 16S rRNA and complementary mRNA Shine-Dalgarno sequences.nslation 8. To add113 Ambrx, Inc., developed an anti-HER2 ADC productARX788 using the UAA incorporation strategy. ARX788 is generated bythe formation of a highly stable oxime bond between a noncleavableAmberstatin (AS269) drug linker and a ketone-bearing UAA, pAcF, whichwas incorporated into the primary sequence of the antibody throughamber codon suppression. The Food and Drug Administration has grantedfast track designation to ARX788 for the treatment of patients withadvanced or metastatic HER2-positive breast cancer in 2021.114The UAAtechnology is ideal for antibody–drug conjugate (ADC) generationand other applications in biomedicine. The problem is production ofhomogeneous titers of ADCs for targeted cancer chemotherapy. ADCsare anticancer therapies designed to target tumors with cytotoxicsmall molecule drugs. Bypassing normal tissues reduces the toxic sideeffects of chemotherapy. Site-specific conjugation of the small moleculedrug to the antibody homogenizes the mixture of ADCs, thus providingmore reliable pharmacokinetic properties, efficacy, and safety profiles.In 2014, Tian and co-workers produced gram per liter scale titersof UAA-incorporated ADCs from stable CHO cells using antibodies targetingcommon antigens on colorectal/gastric and breast cancers.115 In 2016,the Zhou lab developed a live attenuated influenza A vaccine straincontaining multiple amber codons in its genome.116 The strain can be replicated only in a transgenic 293Tcell line that harbors an orthogonal amber suppressor aaRS/tRNACUA pair and the cognate UAA, making it replication incompetentin normal human cells and thus useful for immunization.116 Undoubtedly, the UAA technology can be exploitedin other modalities for therapeutics.UAAs can also be used for vaccine development.In 2014, Wang and co-workers developed a theoretically safe and effectiveHIV-1 vaccine by making viral replication dependent on the presenceof UAA and the aaRS/tRNA pair.in vivo withno cross reactivity, the UAA technology willbe a powerful tool for systems biology that enables biological mechanismto be studied in living cells. As an example, biologists would liketo control various protein signaling states by turning on and offreceptor–ligand interactions and intraprotein interactions.Such control would enable clear-cut conclusions about the functionaleffects of specific protein structures in the native cell environment.This was recently illustrated in a study by the Chen lab exploitingtransition metal-based bio-orthogonal cleavage reactions for on-demandrelease of toxic drugs from ADCs and precise control of ligand–receptorinteractions at the cell surface.117 Inthis study, they used the genetic code expansion strategy to incorporatechemically caged Tyr and Lys analogues into eight different sitesof ZHer2, a small protein with a high affinity for themembrane protein HER2. The UAA-modified ZHer2 mutants wereexpressed in E. coli, purified, and fluorescentlylabeled. Fluorescent ZHer2 and its UAA-modified mutantswere incubated with SK-BR-3 cells, a human breast cancer cell linethat overexpresses HER2, and analyzed by flow cytometry. Strong fluorescencewas observed when wild-type ZHer2 and HER2 interacted.A decrease in fluorescence was observed with caged UAA-modified ZHer2 mutants that could no longer interact. Fluorescence wasrescued upon decaging to the native amino acid. This allowed themto directly probe the functional role of each amino acid residue inthe interaction between ZHer2 and Her2.117 It should be emphasized that the UAA technology is at asufficiently mature stage where it can be adopted by biologists notexpert in the methodology with similar ease and with even more diverseapplications, perhaps, than GFP.Once multiple different UAAscan be incorporated at unique codons Considerable progress has been made in the field of genetic codeexpansion since the publication of the study by Chatterjee et al.in 2013. However, many technical challenges remain, and the applicationof the technology to drug development and basic science is only justbeginning.in vivo, and a growing arsenalof UAAs. Together, these advances allow direct selection for orthogonalribosomes that work with an expanded set of UAAs and codons.Significantly changing the structure of the aminoacid and stillhaving it be accepted as a substrate by the translational machineryremains difficult. For example, there are limitations to what backboneanalogues can be incorporated for unnatural oligomer synthesis orpowerful fluorophores for biophysical studies of proteins. There havebeen incredible advances in our understanding of ribosome structureand function since Schultz, Noren, and co-workers published theirseminal paper in 1989. In collaboration, we demonstrated that thismechanistic knowledge could be exploited to gain insight into howthe translational machinery discriminated the structure and electronicsof the amino acid. More mechanistic work with UAAs is needed. Thismechanistic work can provide insight into the mechanism of translationand guide future engineering efforts. It also is now possible to engineertranslation and the ribosome at a scale not previously possible withGROs, stapled ribosomes E. coli a robust method accessibleto scientists not expert in the field. Biochemists and biologistsare encouraged to adopt the technology and apply it to a variety ofmechanistic questions. With the ability to produce a broad range ofUAA-containing proteins in E. coli in high yieldand advances in bio-orthogonal labeling methods, the field is at anexciting moment to apply the technology to challenging problems indrug discovery and biotechnology. Antibody–drug conjugatesand vaccine development likely are just the start for therapeuticapplications of the technology. Undoubtedly, an important next stepfor the field will be synthesizing unnatural oligomers directly in E. coli.Optimization has made expression and purification of UAA-incorporatedproteins from The next challenge is to develop methodsfor incorporating multiple,different UAAs in response to different codons in live mammalian cells.This would allow mechanistic questions, like the role of differentepigenetic modifications, to be addressed directly in the cell. Technically,this likely will require (1) using GROs so the UAAs are not incorporatedat endogenous codons, (2) engineering tethered ribosomes in mammaliancells, and (3) evolving tethered ribosomes to work with differentUAAs and different codons. If successful, like GFP before it, UAAscould be a powerful tool for studying biological mechanism in livecells, but with a much broader repertoire of chemical functionality.de novo GROs. Looking forward, as the tools continueto progress, UAA mutagenesis no doubt will become an essential toolfor asking fundamental questions in systems biology and will be furtheradapted as a new strategy for drug development.The incorporation of unnatural amino acids by the translationalmachinery using artificial UAA-tRNAs is a wonderfully creative ideabringing together organic chemistry, molecular biology, and syntheticbiology of the translational machinery. It is now a robust field withmany who trained in the technology leading their own exciting advancesfor the tools in the UAA repertoire, in the aaRS/tRNA orthogonal pairs,in moving to higher organisms, and even in tethered orthogonal ribosomesand


In March 2020, Bronx County (NY) saw one of the first U.S. COVID-19 outbreaks. This outbreak coincided with the ongoing Einstein Aging Study (EAS), which involved older adults living in Bronx County completing annual two-week intensive data collection “bursts.” Thus, it serves as a natural experiment to study pre-COVID to early pandemic-related changes in the daily well-being of participants who were at risk both due to their age and their location. We examined within-person change in self-reported negative thoughts, affect, stress, and loneliness from a subsample of 78 EAS participants. Participants’ data from a two-week “burst” of momentary surveys during 2019 were compared with their data from the corresponding timeframe during the early COVID-19 period (February-June 2020). Personality and mild cognitive impairment were examined as predictors of change. Average momentary loneliness significantly increased from 2019 to 2020. Participants with greater neuroticism increased more in thought unpleasantness and depressed feelings. To understand the community context, community distress markers were analyzed using Artificial Intelligence (AI)-based assessments of public Twitter posts from Bronx County during the same periods. These Twitter posts also showed a surge of COVID-related topics at the onset of the Bronx outbreak. Language analysis showed a 2019-2020 increase in Bronx community markers of anxiety, depressivity, and negatively-valenced affect extracted from Twitter. We observed 2019-2020 change in both individuals’ well-being (via intensive reports) and in their communities (via Twitter). Contextualizing these with the increased COVID-19 discussion online suggests that these may reflect common pandemic effects.


Systematic screening improves delirium detection among hospitalized older adults. This poster describes the development and pilot testing of an iOS-based app that incorporates the Ultra-Brief Confusion Assessment Method (UB-CAM), a two-step, delirium detection protocol that combines the UB-2 (2-item screener) and 3D-CAM. Previous work tested a RedCAP-based UB-CAM app in 527 patients with 399 physicians, nurses, and certified nursing assistants (CNAs) showing it can be successfully completed by all three disciplines in 97% of eligible patients in 80 seconds on average with over 85% accuracy relative to a gold standard. To improve accessibility to the clinical setting, our research team now collaborated with a computer scientist to develop and refine an iOS-based UB-CAM app for the iPhone and iPad through iterative “laboratory” testing. The app was piloted by non-clinician, research testers in hospitalized older adults  with dementia ; 64% were assessed to be delirium positive. The app demonstrated preliminary efficiency (90 seconds on average), high acceptability (100% satisfaction of users), and reliability (100% inter-rater). This project underscores the need for close collaboration between researchers, clinicians, and computer scientists with iterative testing of bedside-facing apps prior to testing with patients. Next steps include testing effectiveness in a pragmatic trial with clinician users , integrating the UB-CAM app into the routine hospital care of all older patients. Having rapid, accurate bedside delirium detection has the potential to transform care.


With the success of COVID-19 vaccines, newly created mRNA vaccines against other infectious diseases are beginning to emerge. Here, we review the structural elements required for designing mRNA vaccine constructs for effective in vitro synthetic transcription reactions. The unprecedently speedy development of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was enabled with previous innovations in nucleoside modifications during in vitro transcription and lipid nanoparticle delivery materials of mRNA. Recent updates are briefly described in the status of mRNA vaccines against SARS-CoV-2, influenza virus, and other viral pathogens. Unique features of mRNA vaccine platforms and future perspectives are discussed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the causative agent for the rapidly transmitting pandemic coronavirus disease 2019 (COVID-19) across the globe since the first outbreak in December 2019 ,4. Just It is a miraculous scientific triumph to develop novel mRNA vaccines, licensed first in the United Kingdom and then Canada and the USA, for emergency use authorization for humans in an actual pandemic situation within a year with an unprecedented shorter time and higher efficacy than traditional vaccine platforms. COVID-19 mRNA vaccines are not the whole story of the RNA vaccine field. Scientists and companies had been developing, innovating, and improving RNA technologies for almost three decades prior to COVID-19. In the 1990s, the injection of mRNA into mouse muscle resulted in protein expression , and antHere, we review the structural elements in designing mRNA vaccine constructs, the parameters to be optimized, the current updates of mRNA-based vaccines against viral diseases, and the unique features of mRNA vaccine technology in an era of a pandemic. New perspectives and future applications of mRNA technology are discussed to address the development of novel mRNA vaccines against challenging and recurring viruses and the antigenic variants of viral pathogens.For efficient translation, as detailed in previous review articles ,24, in vmpN) or both first and second nucleotides (cap2: m7GpppNmpNm) . Th. Th43]. The specific motif and internal ribosomal entry site (IRES) can control ribosome binding to enable cap-independent mRNA universal translation even in the cells with eIF4E at a low level, but cap structure is still needed to protect from exonuclease attack ,47. IRESWhile the 5′ UTR promotes the initiation of translation, specific sequences in the 3′UTR contribute to stabilizing intracellular mRNA, ultimately enhancing the duration of protein expression . Longer An ORF of mRNA dictates the primary sequence information of the target protein of interest and higher order RNA structures impacting translation efficacy. Codon usage and RNA secondary structures independently contribute to regulating protein expression, suggesting the difficulty of optimization. Unexpectedly, mRNA coding sequences potentially forming secondary structures were shown to be correlated with highly expressed mRNAs . In addiThe poly-A tail, which is a typical length of 60 to 150 nucleotides, is essential for mRNA stability, translation, and recognition by poly-A binding protein (PABP) that subsequently interacts with the translation initiation complex (eIF4G) to form a loop-like conformation  33]. Tw. Tw33]. Once the DNA template is designed and constructed to contain a promoter, UTR, ORF, and poly-T sequences as described above , mRNA caFor co-transcription of capping, IVT reaction solution is prepared to contain cap analogs in optimized ribonucleoside triphosphate substrates, RNA polymerase, and DNA template. Similar to viral RNAs, the host immune system recognizes IVT unmodified mRNAs as exogenous danger signals, activating pattern recognition receptors such as Toll-like receptors (TLRs) and cytoplasmic RNA sensors. Endosomal TLR7 and TLR8 recognize single-strand RNA as ligands, while TLR3 binds to double-strand RNA molecules ,76,77. TThere are additional studies demonstrating the mechanisms of lowering stimulation of the innate immune system in base-modified IVT mRNA products. Use of base-modified nucleotides led to reduced synthesis of antisense duplex mRNA by-products and yielded less inflammatory mRNA ,87. SecoIVT mRNA transcripts are unstable and highly susceptible to degradation by nucleases as evidenced by a short half-life (<5 min) in sera . Most ceThe mRNA vaccines after intramuscular injection are taken up by antigen-presenting cells via endocytic pathways . The mRNResearch developments and innovations in the use of synthetic mRNA methods and processes for several decades led to the first Phase 1 clinical studies on influenza hemagglutinin (HA) mRNA lipid nanoparticle vaccine in 2015 ,22. A prWith unprecedented speedy preparedness and effort, clinical phase 1 and 2 studies and phase 3 efficacy trials of mRNA vaccines were initiated and carried out approximately 2 and 4 months after sequence availability in the real COVID-19 pandemic situation ,112. TheSelf-amplifying mRNA contains cis or trans RNA transcripts encoding non-structural proteins forming RNA-dependent RNA polymerase (RDRP), as well as mRNA for protein antigens of interest . Owing tBefore COVID-19, mRNA-based influenza vaccines were investigated extensively in preclinical studies due to the ease of testing efficacy in small animal models, which led to the first clinical trials ,22 and pThe development of mRNA-based vaccines against infectious diseases has been previously summarized in self amplifying and conventional non-replicating RNA technologies ,124,125.+ and CD8+ T-cell responses in mice [Nucleoside modified RSV mRNA (m1ψ-mRNA/LNPs) vaccines encoding either prefusion F stabilized or wild type fusion (F) were reported to be effective in inducing neutralizing antibodies in mice (10 μg) and cotton rats (25 μg), as well as CD4 in mice . A phase in mice . All dos in mice .Zika virus (ZIKV) pre-membrane and envelope (prM-E) glycoprotein encoding modified mRNA LNP vaccine provided protection against ZIKV challenge in mice (30 μg mRNA) and in NHPs (50 μg mRNA) after a single dose . SteriliA non-replicating mRNA vaccine encoding rabies virus glycoprotein (Rabies-G) could induce neutralizing antibodies that last for one year, CD4 and CD8 T cell responses, and protect against lethal intracerebral challenge in mice . A thermHIV-1 envelope gp160 encoding nucleoside-modified mRNA LNP vaccination induced polyfunctional HIV-1 antibodies at a similar or superior magnitude and breadth compared to adjuvanted protein immunization and durable neutralizing antibodies for 41 weeks in NHPs . NucleosIt is advised not to administer the COVID-19 mRNA LNP vaccine in subjects with a significant history of allergic reactions because of potential severe adverse effects . PolyethEndogenous reverse transcriptase (RT) activity was shown to serve as a translational repressor in platelets via RNA–DNA hybrids and treatment with RT inhibitors resulted in enhanced global protein synthesis and platelet activation . A recenThe success of mRNA-based COVID-19 vaccines has proven the unique features of mRNA vaccine technology. Numerous conventional and new vaccine platforms competed with one another to develop COVID-19 vaccines right after the sequence information of the new SARS-CoV-2 was available. The mRNA vaccines from Pfizer/BioNTech and Moderna quickly turned out to be the frontrunners, obtaining the emergency use approval for prophylactic COVID-19 vaccination within 11 months. The speed of developing mRNA-based COVID-19 vaccines was much faster than any other vaccine platform, probably owing to the nature of IVT mRNA preparations, which does not require cell culture, biohazard materials, and complex purification procedures. Of course, it is an important lesson that the quicker development of mRNA COVID-19 vaccines resulted from almost three decades of continued research efforts and innovations in mRNA technology. The efficacy (~95%) of protection by mRNA vaccination [Now the mRNA vaccine technology has the versatility of substantial room for fine-tuning molecular design regardless of antigens. The commercial scale-up manufacturing process would stay the same for any other mRNA sequence. The success of mRNA-based COVID-19 vaccines is opening up new avenues of other mRNA vaccines against recurring infectious diseases such as influenza, RSV, rabies, Zika, HIV, herpes, dengue, hepatitis, and malaria. The quicker production of mRNA vaccines will be more responsive to circulating influenza variants and the emergence of unpredictable pandemic viruses than the egg-based lengthy traditional methods. This short timeline and high efficacy in mRNA vaccine technology are particularly advantageous during the outbreaks of new viruses or pandemics. Moderna and BioNTech companies aim to develop several mRNA vaccine candidates targeting pan-coronavirus, influenza, rabies, Zika, and HIV in the developmental pipeline ,125. DelNew mRNA vaccine innovations are expected to replace conventional vaccine platforms and develop more effective vaccines against recurrent and challenging pathogens or cancers in the near future. More development in delivery vehicles of mRNA will be required for safer, effective, and cold-chain-free mRNA vaccines. An example is the development of thermostable mRNA vaccines ,132. FurIntramuscular injection is the common route of administration for currently licensed COVID-19 vaccines, inducing systemic humoral and cell-mediated immune responses and protection against lung infection, inflammation, severe disease, and death. Breakthrough infections can occur in COVID-19 vaccinees that can transmit SARS-CoV-2 to unvaccinated individuals. Intranasal dosing of ChAd-SARS-CoV-2-S adenoviral based vaccine was shown to induce sterilizing immunity preventing SARS-CoV-2 infection in both the upper and lower respiratory tracts in mice ,146. An