The growing understanding of RNA functions and their crucial roles in diseases promotes the application of various RNAs to selectively function on hitherto “undruggable” proteins, transcripts and genes, thus potentially broadening the therapeutic targets. Several RNA-based medications have been approved for clinical use, while others are still under investigation or preclinical trials. Various techniques have been explored to promote RNA intracellular trafficking and metabolic stability, despite significant challenges in developing RNA-based therapeutics. In this review, the mechanisms of action, challenges, solutions, and clinical application of RNA-based therapeutics have been comprehensively summarized. Small molecules and antibody drugs target only 0.05% of the human genome, and most disease targets lack defining active sites for small-molecule binding.Abundant RNAs selectively act on proteins, transcripts and genes, which broaden the range of druggable targets. A defined sequence of RNA drugs makes the design of RNA therapeutics much easier.Multiple RNA drugs are approved and a dozen more are in phase III trials to treat rare and common diseases.How do RNA molecules function in treating diseases?The challenges and solutions to the discovery and development of RNA-based drugs effectively.The antisense oligonucleotides (ASOs) inhibiting protein synthesis in the early 1980s promoted the rapid advances of RNA-based therapeutics . Other RCompared to conventional protein-targeted and DNA-based medicines, RNA-based therapeutics are prospective due to their distinct physicochemical and physiological properties . RNAs fuThis review provides an overview of significant developments in RNA-based therapeutics. The classification of RNA-based therapies and their modes of action have been outlined. This review also covers critical challenges in applying these RNA therapies and possible solutions. Finally, the current preclinical and clinical trials of RNA therapeutics have also been summarized.The ASOs are short single-stranded (ss) oligonucleotides (12–24 nucleotides (nt)) complementary to the specific RNA through Watson–Crick base-pairing. They can alter RNA and reduce, restore, or modify protein expression . ASOs arIn the occupancy-mediated degradation, ASOs bind to and cleave the target RNA at the ASO binding sites through endogenous enzymes, enhancing the downregulation of target transcripts Fig. . This meIn the occupancy-only mechanisms, ASOs control the down-/ up-regulation of target transcripts by binding to them without the help of specific enzymes. Sometimes this mechanism is also called the steric block mechanism. Altering RNA splicing is the most widely used strategy . Splice-RNAi is an endogenous cellular process inducing double-stranded (ds) RNAs -triggered degradation of particular RNA targets, which provide an intrinsic defensive mechanism against invading viruses and transposable elements , 27, 28.amyloid-β peptide precursor protein (APP) mRNA 5′-UTR to upregulate APP translation and amyloid-β production [IL-10 mRNA stability and IL-10 protein expression through binding to IL-10 AU-rich elements [miRNAs are endogenous single-stranded small ncRNAs influencing gene expression via RNAi . The biooduction . miR-466elements . miRNAs elements .Unlike RNAi, RNA activation (RNAa) is a process where dsRNA triggers gene production by targeting promoter sequences . Small aStreptococcus pyogenes (RCas9) requires a matching gRNA and complementary PAM-presenting oligonucleotide (PAMmer) [Campylobacter jejuni and Staphylococcus aureus) can cleave ssRNA without PAM [DD) [The prokaryote-derived CRISPR-associated protein (Cas) systems have been widely used in mammalian cells and organisms to precisely edit genome sequence, resulting in irreversible knockout or knockin of a target gene . Mechani(PAMmer)    ) can also improve delivery. GalNAc is a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), thereby significantly enhancing ASO and siRNA delivery to the liver [Chemical modification of the phosphate backbone, ribose ring, and 3′- and 5′-terminals can improve substrate specificity, nuclease resistance and delivery. Besides, it reduces toxicity and immunogenicity . The modhe liver   tail . The optDespite their robustness and simplicity, therapeutic use of CRISPR systems has many obstacles, including effective delivery, detectable off-target effects, potential immunogenicity, etc. Chemical modification patterns used in ASOs and siRNAs can be applied to gRNA and Cas9 mRNA . For insLike ASOs and siRNAs, versatile chemical modifications and conjugations can enhance the pharmacokinetic features of RNA aptamers . Most apDeveloping an effective carrier to protect the carried RNA from the harmful physiological environment is necessary since RNAs have substantial negative charges and chemical modifications. Advances in nanotechnology and materials science offer advantages and potential solutions to the challenges of oligonucleotide drug delivery, especially for the requirements of intracellular delivery across biological barriers and membranes. Key benefits of nanoparticle drug delivery systems include custom optimization of nanoparticle biophysics  and biological properties , allowing for a high degree of customization delivery platform. Here we introduced two dominant delivery approaches—lipid nanoparticles and cationic polymer-based polyplexes, and three emerging novel nanocarriers. We summarize the representation of RNA therapeutics delivery as shown in Table LNPs are the most widely used carriers to deliver oligonucleotide drugs , 87. LNPUsing the LNP-mediated siRNA delivery as an example  Fig. 3B3B. The aribonucleotide reductase M2 mRNA in patients with solid cancers [Polyplexes are standard formulations used for nucleic acid delivery. They are spontaneously formed by electrostatic interactions between cationic polymers and negatively-charged nucleic acids Fig. . The pol cancers , 101. Ot cancers  and poly cancers  are alsoExosomes are lipid membrane-enclosed vesicles with a diameter ranging from 40 to 160 nm Fig. . They arTNF-α mRNA via ASOs. XCUR17 can target interleukin-17 receptor-α through ASOs for treating psoriasis micro plaques. ST-008, TLR9 agonist SNA, combined with immune checkpoint inhibitors, are used for immuno-oncology treatments [SNAs are densely packed, radially oriented nucleic acids in the outer layer and covalently bond to an inorganic nanoparticle core. Gold nanoparticles (10–15 nm) are the most commonly used SNA cores. Alkyl thiol or cyclic disulfide chemical tethering groups are used to attach nucleic acids to the core surface. A recognition sequence (15–25 nt) is complementary to the target sequence, and a spacer (10 nt) connects the nanoparticle surface with the recognition sequence  Fig. 3B. SNAs caeatments .DNA-based nanostructures are also widely used as delivery vehicles Fig. . The selRNA-based therapeutics were firstly adopted for diseases with clear rationales, such as oncology, neurological disorders and infectious diseases. Recent advancements in RNA technologies have led to the approval of eight ASOs, three siRNAs, and two mRNA vaccines for COVID-19 under the Emergency Use Authorization scheme Table . BesidesOut of the 100 phase I trials performed on ASO-based therapies, a quarter has entered phase II/III trials in the last 5 years for treating rare and common diseases, such as orphan genetic alterations and cancer .apolipoprotein B (ApoB) mRNA. Likewise, inotersen mediates RNase H1-mediated degradation of hepatic transthyretin (TTR) mRNAs to reduce TTR protein synthesis and serum TTR levels [Fomivirsen is the first FDA-approved ASO drug for treating cytomegalovirus retinitis (CMV) in patients with AIDS . This PSR levels . Two splR levels . NusinerR levels . EteplirR levels . Since eR levels , viltolaR levels , and casR levels  have beeR levels .vascular endothelial growth factor receptor one (VEGFR1) mRNA. However, the poor efficacy prevents its clinical development [Several clinical trials are being conducted to test the efficacy of ribozymes in the treatment of solid tumors, HIV and other diseases . RPI.461elopment . OZ1 is elopment . Althougelopment .Three siRNA drugs  have been approved by FDA to date, while seven siRNA candidates  are undergoing Phase III clinical trials.G12D siRNA (siG12D) drug for the treatment of pancreatic ductal adenocarcinoma (NCT01188785) [Patisiran is the first FDA-approved RNAi-based drug for treating hTTR with polyneuropathy, ushering in a booming new era for RNAi therapeutics . Similar1188785) . Additio1188785) .miRNA inhibitors (Anti-miRs) and miRNA mimics can be used to down- or upregulate miRNAs. Miravirsen (SPC3649) and RG-101are anti-miRs targeting miR-122 for treating hepatitis C virus infection . The miRMTL-CEBPA is the first saRNA in clinical trials. It regulates hepatic and myeloid functions, as well as numerous oncogenic processes by upregulating the transcription factor CCAAT/enhancer-binding protein alpha (C/EBP-α) , 141. ThThe first human clinical trial on applying CRISPR/Cas gene editing involved using ex vivo Cas9 to knockout PD-1 in autologous T cells (NCT03399448) . In addiPegaptanib (Macugen) is the first FDA-approval aptamer drug targeting VEGF to treat age-related macular degeneration . NumerouIn the last few decades, mRNA vaccines have gained widespread applications. In 2020, the outbreak of coronavirus disease 2019 (COVID-19) stimulated the most rapid development of mRNA vaccines in history.®) [Vaccines targeting infectious diseases represent the most advanced application of mRNA therapies. So far, mRNA vaccines for various infectious diseases, including influenza, Zika, and respiratory syncytial virus, have been developed . During ®)  and the ®)  were the®) .The application of mRNA-based cancer vaccines has been recently reviewed , 151. To3- to 106-fold higher than reported chemical compounds (C16 and 2-AP) [Apart from miRNA antagomir and miRNA mimic, artificial circular RNA (circRNA) sponges (circmiRs) are promising therapeutic miRNA antagonists. circRNAs are a subclass of ncRNAs that exist as continuous loop RNAs due to the lack of free 3′ and 5′ ends. They are resistant to nuclease degradation and more stable than linear RNAs. The most frequently described function of circRNAs is acting as miRNA sponges. These circRNAs contain multiple miRNA-binding sites for miRNAs binding, preventing the interaction between miRNA and their canonical mRNA target gene , 153. Rend 2-AP) . Moreovend 2-AP) . In factnd 2-AP) –159.Taken research advance of Cas13d mediated—genome therapy as another example. Cas13d has been shown to efficiently down-regulating cellular RNAs in mammalian cells in vitro . The adeVarious RNA-based approaches have been applied to experiments and clinical trials. Commoditized ASOs, siRNAs, antagomirs and aptamers are widely used for cell and animal experiments. Several ASOs, siRNAs, aptamer and mRNA vaccines have been approved for clinical application. These approaches allow for the down- or up-regulation of specific mRNA expression and inhibition of ncRNA functions by targeting particular RNA sequences. However, the most significant obstacle preventing the widespread usage of RNA-based approaches is the difficulty of efficiently delivering such drugs to target organs and tissues apart from the liver. In addition, off-target binding , sequencChemical modification is one of the most promising strategies for delivering RNA-based drugs. Modification of the nucleic acid backbone, ribose ring, and nucleobase itself has been widely used to optimize drug-like characteristics for enhanced delivery. For example, extensive chemical modifications allow the delivery of gapmer ASOs to various tissues without an extra delivery agent. To date, eight of the ten approved oligonucleotide treatments are applied without an additional delivery vehicle. However, some unnatural nucleotides may be harmful. For instance, LNA-modified nucleic acids were found to cause severe hepatotoxicity . TherefoDeveloping nanocarriers for RNA drug delivery provides excellent hepatic transport. Non-liver systemically administered nanomedicines require further investigation. Advances in imaging techniques   and omicSMN2 pre-messenger RNA splicing. It is synthesized efficiently and can be easily administered [Apart from the sequence-based method, small-molecule-based therapy to target RNA would be preferred in many cases . Evrysdinistered . Tutorianistered , 170.In summary, combining RNA chemical modifications and conjugation with nanocarrier systems can improve the efficiency of RNA drug delivery. Further research on RNA-based therapeutics, including RNA molecules as therapeutic drugs and targeting RNA with small molecules, will lead to more RNA-based therapeutics for patient treatment.


We further demonstrate that amino acid-bridgedRNAs can act as templates in a subsequent round of copying. Our resultssuggest that aminoacylation facilitated non-enzymatic RNA replication,thus outlining a potentially primordial functional link between aminoacylationchemistry and RNA replication.Aminoacylated tRNAsare the substrates for ribosomal protein synthesisin all branches of life, implying an ancient origin for aminoacylationchemistry. In the 1970s, Orgel and colleagues reported potentiallyprebiotic routes to aminoacylated nucleotides and their RNA-templatedcondensation to form amino acid-bridged dinucleotides. However, itis unclear whether such reactions would have aided or impeded non-enzymaticRNA replication. Determining whether aminoacylated RNAs could havebeen advantageous in evolution prior to the emergence of protein synthesisremains a key challenge. We therefore tested the ability of aminoacylatedRNA to participate in both templated primer extension and ligationreactions. We find that at low magnesium concentrations that favorfatty acid-based protocells, these reactions proceed orders of magnitudemore rapidly than when initiated from the  In extantbiochemistry, theaminoacylation of RNA generates activated tRNA substrates for proteinbiosynthesis. Explaining how RNA could be aminoacylated without enzymes,and how such aminoacylation might have benefitted early protocells,may help to explain how the RNA World underwent the transition toprotein-centric biology. The covalent linkage of amino acids to RNAwould have unavoidably affected RNA replication. If these effectswere beneficial, then efficient ribozyme catalyzed aminoacylationcould have evolved. Once in place, ribozyme catalysis of aminoacylationcould in turn have led to other uses for covalently attached aminoacids, such as peptide formation.1  in the presence of CO2.4 NCAs are inefficient reagents for the direct aminoacylationof ribonucleotides.5 High-yielding aminoacylationpathways employing NCAs6 or in situ activationchemistry7 are known, but they requirea 3′-phosphate moiety and are generally limited to N-blockedamino acids. Therefore, we still do not have a high-yielding and prebioticallyplausible means of chemically aminoacylating RNA strands terminatingin a vicinal diol. However, even inefficient chemistry could havehad significant effects on RNA replication and assembly processes.Nucleotides and RNA strandscan be aminoacylated at the 2′(3′)-hydroxylgroups by reaction with amino acid imidazolides1 1A, which8 On a poly(U) template,2′(3′)-glycyl adenosine reacted with 5′-imidazole-activatedAMP to afford a dinucleotide species that was bridged by the glycineresidue.8 Formation of such phosphoramidate-linkedamino acid RNA “copolymers” dramatically increases thestability of both the aminoacyl ester and phosphoramidate bonds, suggestinga mechanism for enhanced covalent capture of amino acids by earlyRNA.9 In parallel, the Orgel laboratory11 and later our own group14 demonstrated that nucleotideswith either 2′- or 3′-amino groups exhibit a large increasein the rate and yield of non-enzymatic polymerization due to the greaternucleophilicity of the amine substituent relative to that of a hydroxylgroup. These results have been extended to ligation reactions by theKrishnamurthy group15 and our own recentassembly of an active ribozyme via a series of 3′N–5′Pligation reactions.17 While the α-amino groupof aminoacylated RNA should be more nucleophilic than the hydroxylgroup of RNA, it was unclear whether the different location of theα-amino group or the increased steric bulk in the reaction centerwould diminish the rate of primer extension or ligation reactions.Indeed, replacing the 3′-hydroxyl group with a hydroxymethylgroup completely abolished reactivity.18 Together, these results inspired us to test whether the reactionof the free amino group of 2′(3′)-aminoacylated RNAwith phosphorimidazolide-activated nucleotides would result in enhancedrates of RNA copying, and whether such amino acid-bridged oligonucleotidescould act as templates for cycles of replication -aminoacylatednucleotides can react with nucleotide 5′-phosphorimidazolidesin a template-directed process to form phosphoramidate-linked productsvia transamidation.lication 1B.Here, we report thatterminal 2′(3′)-aminoacylationof RNA can indeed enhance both template-directed primer extensionand ligation reactions with imidazole-activated downstream speciesby >2 orders of magnitude. We also show that RNA oligomers containinga single amino acid “bridge” can act as templates forsubsequent rounds of RNA template copying. Taken together, our findingsreveal a potential pathway by which non-enzymatic RNA aminoacylationcould have facilitated non-enzymatic RNA replication. The subsequentevolution of a more efficient ribozyme-catalyzed aminoacylating activitycould thus have been directly advantageous for early protocells, whileat the same time generating the aminoacylated RNA substrates requiredfor the later evolution of ribozyme-mediated peptide synthesis.19 Polymerase chain reaction was performed withHot Start Taq 2X Master Mix, and in vitro transcription with the HiScribeT7 Quick High Yield RNA Synthesis Kit from New England Biolabs . EDTA is used as an abbreviation for Na2EDTA (pH 8.0).All reagents were purchased fromSigma-Aldrich  unless specified otherwise. TurboDNasewas purchased from Thermo Scientific . Flexizyme “dFx”and the corresponding mutant M2 were prepared as described elsewhere.Oligonucleotides were eitherpurchased from Integrated DNA Technologies  or synthesizedin house on an Expedite 8909 solid-phase oligo synthesizer. Phosphoramiditesand reagents for the Expedite synthesizer were purchased from eitherGlen Research  or Chemgenes . Cleavageof synthesized oligonucleotides from the solid support was performedusing 1 mL of AMA (1:1 mixture of 28% aqueous ammonium hydroxide and40% aqueous methylamine) for 30 min at room temperature, while deprotectionwas performed in the same solution for 20 min at 65 °C. Deprotectedoligos were lyophilized, resuspended in 100 μL of DMSO and 125μL of TEA·3HF, and heated at 65 °C for 2.5 h to removeTBDMS from 2′-hydroxyls. Following this deprotection, oligoswere purified by preparative 20% polyacrylamide gel electrophoresis(19:1 with 7 M urea), desalted using Waters  Sep-PakC18 cartridges, and characterized by high-resolution mass spectrometryon an Agilent 6230 TOF mass spectrometer.20 with the following modifications. Gel-purified products of 1 μmolsolid-phase synthesis were dissolved in 100 μL of DMSO; 0.05mmol of triethylamine (TEA), 0.02 mmol of triphenylphosphine (TPP),0.04 mmol of 2-methylimidazole (or 2-aminoimidazole), and 0.02 mmolof 2,2′-dipyridyldisulfide (DPDS) were added to the reactionmixture, and the reaction mixture was incubated on a rotator for 5h at room temperature. After 5 h, all of the reagents mentioned abovewere added in listed quantities again and the reaction mixture wasallowed to rotate for an additional 12 h at room temperature. Thereaction mixture was precipitated with 100 μL of saturated NaClO4 in acetone and 1 mL of acetone for 30 min on dry ice. Thepellet was washed twice with 1 mL of a 1:1 acetone/diethyl ether mixture.The products were resolved and purified by HPLC on an Agilent ZORBAXanalytical column , at a flow rate of 1 mL/min. The followinggradient was used: (A) aqueous 20 mM triethylammonium bicarbonate(pH 8.0) and (B) acetonitrile, from 7% to 12% B over 12 min.Oligonucleotidesphosphorylated on the 5′-OH were activated with 2-methylimidazoleor 2-aminoimidazole as previously reported2-Aminoimidazolium Cytidine Dinucleotide (C*C).First, 0.46 mmol of CMP (free acid) was dissolved in 4 mL of DMSO,and 2.9 mmol of TEA, 3.8 mmol of TPP, and 0.22 mmol of 2-aminoimidazole were added to the CMP solution. The resulting suspensionwas sonicated and heated briefly until all of the reagents had completelydissolved. Four millimoles of DPDS was added to the solution to startthe reaction, and the reaction mixture was stirred for 15 min at roomtemperature. The reaction mixture was then precipitated by adding0.5 mL of saturated NaClO4 in acetone and 60 mL of a 1:1acetone/diethyl ether mixture, washed twice with a 1:1 acetone/diethylether mixture, and purified by C18 reverse-phase chromatographyat a flow rate of 40 mL/min. The following gradient was used: (A)aqueous 2 mM triethylammonium bicarbonate (pH 8.0) and (B) acetonitrile,from 0% to 10% B over 10 min.2-Aminoimidazolium Guanosine-UridineDinucleotide (G*U). For OAt-GMP synthesis, 0.275 mmol ofGMP (free acid) was dissolvedin 18 mL of water, followed by 1.47 mmol of HOAt and 1.5 mmol of TEA.The solution was then lyophilized. The resulting powder was dissolvedin 10 mL of DMSO, followed by the addition of 3.6 mmol of TEA, 2.75mmol of TPP, and 2.75 mmol of DPDS. The reaction mixture was stirredat room temperature for 30 min, precipitated by adding 0.5 mL of saturatedNaClO4 in acetone and 60 mL of a 1:1 acetone/diethyl ethermixture, washed twice with a 1:1 acetone/diethyl ether mixture, andpurified by C18 reverse-phase chromatography at a flowrate of 40 mL/min. The folowing gradient was used: (A) aqueous 2 mMtriethylammonium bicarbonate (pH 8.0) and (B) acetonitrile, from 0%to 15% B over 10 min. For 2-AI-UMP synthesis, UMP was activated asC*C, except 1.38 mmol of 2-aminoimidazole was added (3 equiv). PurifiedOAt-GMP and 2-AI-UMP were then mixed in 4 mL of water for 1 h. G*Uwas purified by preparative HPLC on an Agilent preparative column, at a flow rate of 15 mL/min. The following gradientwas used: (A) aqueous 2 mM triethylammonium bicarbonate (pH 8.0) and(B) acetonitrile, from 2% to 8% B over 20 column volumes.19 with the following modification. N-Boc-protected amino acid DBE-esters were deprotected in2 mL of neat TFA for 10 min, followed by washing with 10 mL of 3×diethyl ether. Products were obtained as TFA salts. TFA salts weredissolved in 100% DMSO to a final concentration of 25 mM and usedin reactions directly. 1H NMR spectra were recorded usinga 400 MHz NMR spectrometer (Varian INOVA) operating at 400 MHz. Low-resolutionmass spectrometry was performed by directly injecting 10 μLof a 2 mg/mL solution in 1:1 acetonitrile/water mixture on an Esquire6000 mass spectrometer . High-resolution mass spectrometrywas performed by injecting 500 pmol of material dissolved in wateron an Agilent 1200 HPLC instrument coupled to an Agilent 6230 TOFmass spectrometer.Amino acid-DBE substrates were synthesized as reportedpreviously19 with the following modifications. A typical 10 μL reactionmixture contained 50 mM Na-HEPES (pH 8.0), 10 mM MgCl2,10 μM fluorescein-labeled primer, 5 mM aa-DBE , and 10 μM dFx Flexizyme. The reaction mixture was incubatedon ice for 12–16 h. One microliter of the reaction mixturewas quenched with 9 μL of quench buffer  and loaded intoa 20% polyacrylamide gel [19:1 with 7 M urea and 0.1 M NaOAc (pH 5.0)]in a cold room (4 °C). The gel was run for 2 h at 300 V and visualizedon a Typhoon 9410 imager. A typical aminoacylation reaction yielded30–60% product, measured by band densities in ImageQuant TLsoftware.Aminoacylation reactionswere performed as reported previously1) was synthesizedas described previously21 with the followingmodifications. Oligonucleotide aminoacylation was performed at a 1mL scale and then split into two followed by the addition of the FX_T2hybrid template and FX_S2 “sandwich” (Table S1) to final concentrations of 2.5 μM each. Na-HEPES(pH 8.0) and EDTA were added to final concentrations of 200 and 50mM, respectively. The solution was allowed to warm to room temperaturefor 2 min, after which the reaction was started by the addition ofthe C*C dinucleotide to a final concentration of 13.5 mM. The reactionwas allowed to proceed for 10 min while the mixture was being rotatedat room temperature. The reaction mixture was then concentrated usingAmicon Ultra-4 mL 3K centrifugal filters, and the buffer was exchangedtwice with nuclease-free water. The reaction mixtures were combinedand further concentrated to 50 μL with Amicon Ultra-0.5 mL 3Kcentrifugal filters; 375 μL of nuclease-free water, 50 μLof 10× TurboDNase buffer, and 50 units of 2 units/μL TurboDNasewere added. TurboDNase digestion was allowed to proceed for 15 minat 37 °C. The digested reaction mixture was then concentratedusing Amicon Ultra-0.5 mL 3K centrifugal filters, diluted with 5 mMEDTA in 95% (v/v) formamide, and purified by preparative 20% polyacrylamidegel electrophoresis (19:1 with 7 M urea) at 4 °C. The desiredgel band was cut out, crushed, and extracted with 1 mL of 50 mM NaOAc(pH 5.5)/50 mM EDTA buffer on a rotator at 4 °C for 16 h. Theextracted product was concentrated using Amicon Ultra-0.5 mL 3K centrifugalfilters, and the buffer was exchanged thrice with nuclease-free waterto yield 80–90% pure 1.A primer with the amino acid bridge beforethe 3′-terminal nucleotide (2) was synthesized byperforming a ligation reaction with the following modifications. Oligonucleotideaminoacylation was performed at a 1 mL scale and then split into twofollowed by the addition of the NP DNA T template (Table S1) to a final concentration of 2.5 μM. Na-HEPES(pH 8.0) and EDTA were added to final concentrations of 200 and 50mM, respectively. The solution was allowed to warm to room temperaturefor 2 min, after which the reaction was started by the addition of2-methylimidazole-activated Ligator1 (Table S1) to a final concentration of 10 μM. The reaction was allowedto proceed for 1 h while the mixture was being rotated at room temperature.The reaction mixture was concentrated with Amicon Ultra-4 mL 3K centrifugalfilters, and the buffer was exchanged twice with nuclease-free water.The reaction mixture was further concentrated to 50 μL usingAmicon Ultra-0.5 mL 3K centrifugal filters. DNA digestion and purificationwere performed exactly as described for 1. After gelextraction and buffer exchange, the 90% pure 2 was precipitatedwith 0.1 volume of 5 M NH4OAc and 3 volumes of isopropanol.A template withan internal amino acid bridge , and 20 mM C*C. Reactions were performedin technical triplicates. At indicated time points, 1 μL ofeach reaction was quenched with 29 μL of quench buffer . Prior to being loaded on20% polyacrylamide gels (19:1 with 7 M urea), the quenched reactionmixtures were heated at 92 °C for 2 min to denature the duplex.Aliquots (3 μL) were loaded into gels and run at 20 W for 80min. The gels were imaged on a Typhoon 9410 imager, and band densitiesquantified in ImageQuant TL software.The RNA template and the downstream RNA oligonucleotide(“sandwich”) were added to a typical 10 μL aminoacylationreaction mixture to final concentrations of 3.75 and 2.5 μM,respectively, followed by Na-HEPES (pH 8.0) to a final concentrationof 200 mM. MgClTo independently determinethe kinetics of pure RNA primer extension, primer extension was performedwith the non-aminoacylated RNA primer. The non-aminoacylated primerwas subjected to typical aminoacylation conditions, except dFx Flexizymewas replaced with water for those reactions. The primer extensionreactions were then set up exactly as in the preceding paragraph.The rate of hydrolysis of the aminoacylated primer was measuredunder primer extension conditions except that CMP was used insteadof C*C. The hydrolysis reaction was quenched using the acidic quenchbuffer , themixture heated at 92 °C for 2 min to denature the duplex, andthe reaction run on an acidic 20% polyacrylamide gel [19:1 with 7M urea and 0.1 M NaOAc (pH 5.0)].1 and the downstream“sandwich” were annealed to the RNA template in a solutioncontaining 3.6 μM 1, 3.6 μM “sandwich”,5.4 μM template, 50 mM Na-HEPES (pH 7.5), 50 mM NaCl, and 1mM EDTA by being heated for 3 min at 70 °C and slowly cooledto 20 °C at a rate of 0.1 °C/s. The annealed solution wasdiluted with Na-HEPES (pH 8.0) and MgCl2, before the reactionwas initiated by adding the C*C dinucleotide. The final reaction concentrationswere 0.6 μM 1, 200 mM Na-HEPES (pH 8.0), 50 mMMgCl2, and 20 mM C*C. The reaction was quenched at theindicated time points, subjected to gel electrophoresis, and quantifiedas described above.The purified product 2 or the all-RNA template) in a solution containing 3.6 μMprimer, 3.6 μM “sandwich”, 5 μM template,50 mM Na-HEPES (pH 7.5), 50 mM NaCl, and 1 mM EDTA by being heatedfor 3 min at 70 °C and slowly cooled to 20 °C at a rateof 0.1 °C/s. The annealed solution was diluted with Na-HEPES(pH 8.0) and MgCl2, before the reaction was initiated byadding the G*U dinucleotide. The final reaction concentrations were0.6 μM primer, 200 mM Na-HEPES (pH 8.0), 100 mM MgCl2, and 20 mM G*U. The reaction was quenched at the indicated timepoints, subjected to gel electrophoresis, and quantified as describedabove.The primers and the corresponding downstream“sandwich”oligonucleotides were annealed to the template (either the glycine-linked P) at each time point,starting from the initial fraction of primer (P0), was plotted as −ln(P/P0) versus reaction time, and the observed rate constant, kobs, was estimated by the slope of a linearregression line. This kobs correspondedto k3 in the kinetic model used to obtainthe kobs(k1) of the aminoacylated primer.For the RNA reaction, primerextension was quantified for each time point by integrating the bandintensity in each gel lane. The band intensity was normalized in eachlane. The remaining primer (P) at each time point,starting from the initial fraction of primer-gly (P0), was plotted as −ln(P/P0) versus reaction time, and the observed rateconstant, kobs, was estimated by the slopeof a linear regression line. This kobs corresponded to k2 in the kinetic modelused to obtain the kobs(k1) of the aminoacylated primer.For the hydrolysis reaction,hydrolysis was quantified for each time point by integrating the bandintensity in each gel lane. The band intensity was normalized in eachlane. The remaining primer-gly With the C*C Dinucleotide  at each time point, starting from the initial fraction of primer(P0), was plotted as −ln(P/P0) versus reaction time,and the observed rate constant, kobs,was estimated by the slope of a linear regression line.Figure S12)With the G*U Dinucleotide  formamide]. Two microliters of 1.5 M NaOH was added to 24μL of the quenched reaction mixture, which increased the pHof the quenched primer extension reaction mixture to 12. The alkalinereaction mixture was incubated at room temperature for 30 s beforebeing neutralized with 1 μL of 0.5 M HCl to pH 8. Aliquots (3μL) were subjected to 20% polyacrylamide gel electrophoresis(19:1 with 7 M urea) and imaged as the aforementioned primer extensionreactions.A primerextension reaction performedat 2.5 mM MgCl4OAcand 3 volumes of isopropanol on dry ice for 20 min and pelleted at15000 rpm for 15 min at 4 °C. The pellet was washed twice with80% ethanol and resuspended in 21 μL of nuclease-free water.Ten microliters of the precipitated reaction mixture was subjectedto the primer extension procedure described in the preceding paragraphs.Because MgCl2 from the aminoacylation reaction was washedaway during precipitation, MgCl2 was added to a final concentrationof 2.5 mM in the final reaction. In addition, exact primer concentrationsafter precipitation could not be accurately determined due to thepresence of dFx Flexizyme. Primer concentrations were estimated onthe basis of a mock aminoacylation reaction from which dFx Flexizymewas omitted. The mock aminoacylation reaction mixture was subjectedto sulfo-NHS-acetate labeling as described above and used as the “RNA”control.Four microlitersof 180 mM sulfo-NHS-acetate was added to the mixture for a typicalaminoacylation reaction performed at a 40 μL scale, and thereaction mixture was incubated for 2 h at room temperature. The reactionmixture was precipitated with 0.1 volume of 5 M NHPrimerextension reactions were performed as described in the preceding paragraphs.At the indicated time points, 1 μL of the reaction was quenchedin 14 μL of freshly prepared NHS-biotin buffer  and incubated atroom temperature for 1 h to allow for biotin labeling. At 1 h, 15μL of quench buffer was added to each labeling reaction mixture. The quenched reactionmixtures were heated at 92 °C for 2 min, and 3 μL aliquotswere loaded into 20% polyacrylamide gels (19:1 with 7 M urea). Thegels were run at 20 W for 80 min, imaged, and quantified as the aforementionedprimer extension reactions.Table S1), which recognizes the 3′-terminalACA sequences. The RNA template was added to a typical 10 μLaminoacylation reaction mixture to a final concentration of 3.75 μM,followed by Na-HEPES (pH 8.0) to a final concentration of 200 mM.Note that because aminoacylation reactions were performed in the presenceof 10 mM MgCl2, they contributed 2.5 mM MgCl2 to the final reaction. The reaction mixtures were allowed to warmto room temperature for 2 min before the reactions were initiatedby the addition of the 2-methylimidazole-activated decamer [Ligator1(Table S1)] to a final concentration of10 μM. The final reaction concentrations were 2.5 μM mixtureof primers, 2.5 mM MgCl2, 200 mM HEPES (pH 8.0), and 10μM activated decamer. Reactions were performed in technicaltriplicates. At the indicated time points, 1 μL of each reactionwas quenched with 29 μL of quench buffer . Prior to being loaded on 20% polyacrylamidegels (19:1 with 7 M urea), the quenched reaction mixtures were heatedat 92 °C for 2 min to denature the duplex. Aliquots (3 μL)were loaded into gels and run at 20 W for 80 min. The gels were imagedon a Typhoon 9410 imager, and band densities quantified in ImageQuantTL software.Aminoacylation reactions that were used in subsequentligation experiments were performed with dFx Flexizyme mutant M2 .To independently determine the kinetics of pureRNA ligation, ligation reactions were performed with the non-aminoacylatedRNA primer. The non-aminoacylated primer was subjected to typicalaminoacylation conditions, except dFx Flexizyme M2 was replaced withwater for those reactions. The ligation reactions were then set upexactly as in the preceding paragraph. Note that to control for possibleeffects of the different amino acid-DBE esters in the RNA ligationreactions, RNA control reactions were performed in the presence ofeach tested amino acid-DBE ester , 150 mM HCl, 2 μM reverse complementof the template, and 70% (v/v) formamide], heated at 92 °C for2 min to denature the duplex, and run on an acidic 20% polyacrylamidegel [19:1 with 7 M urea and 0.1 M NaOAc (pH 5.0)].P) at each timepoint, starting from the initial fraction of primer (P0), was plotted as −ln(P/P0) versus reaction time, and the observed rateconstant, kobs, was estimated by the slopeof a linear regression line. This kobs corresponds to k3 in the kinetic modelused to obtain the kobs(k1) of the aminoacylated primer.For the RNAreaction, primer extension was quantified for each time point by integratingthe band intensity in each gel lane. The band intensity was normalizedin each lane. The remaining primer (P) at eachtime point, starting from the initial fraction of primer-gly (P0), was plotted as −ln(P/P0) versus reaction time, and the observedrate constant, kobs, was estimated bythe slope of a linear regression line. This kobs corresponded to k2 in the kineticmodel used to obtain the kobs(k1) of the aminoacylated primer.For the hydrolysisreaction, hydrolysis was quantified for each time point by integratingthe band intensity in each gel lane. The band intensity was normalizedin each lane. The remaining primer-gly . The hydrolysis reaction conditions included 0.375 μMoligonucleotide (1 or 2), 200 mM Na-HEPES(pH 8.0), 2.5 or 100 mM MgCl2, and 22 °C (thermocycler).The reactions were stopped by the addition of quench buffer , and the mixtures flash-frozenin liquid nitrogen. The double-stranded quenched reaction mixtureswere heated for 2 min at 92 °C to denature the duplex beforebeing loaded into the gel (the single-stranded reaction mixtures werenot heated).Purified products Kinetic Analysis. Hydrolysis wasquantified for each time point by integrating the band intensity ineach gel lane. The band intensity was normalized in each lane. Theremaining amino acid-bridged oligonucleotide  at each time point, startingfrom the initial fraction of the amino acid-bridged oligonucleotide, was plotted as −ln(P/P0) versus reaction time, and the observed rate constant, kobs, was estimated by the slope of a linearregression line. The half-lives were calculated from the kobs values by the following equation for a first-orderprocess: t1/2 = ln(2)/kobs.2Characterization of the DegradationProduct of . The reaction was performed for24 h in a solution thatcontained 0.375 μM 2, 200 mM Na-HEPES (pH 8.0),and 2.5 mM MgCl2 at 22 °C. After 24 h, the reactionmixture was precipitated with 0.1 V 5 M NH4OAc and 3 Visopropanol, pelleted at 15000 rpm and 4 °C, washed twice with80% ethanol, desalted using a C18 Zip-tip column, and analyzed onan Agilent 1200 HPLC instrument coupled to an Agilent 6230 TOF massspectrometer.in vitro selectionto aminoacylate any tRNA of interest with a wide variety of aminoacids.24 dFx Flexizyme can aminoacylate any RNA sequencethat ends in the CA-3′ sequence found in tRNAs.24 Although aminoacylation occurs at the 3′-hydroxyl,rapid transacylation yields a dynamic mixture of 2′- and 3′-aminoacylatedregioisomers.25 We designed a 10-nucleotideprimer terminating in CCA and tested it for aminoacylation using dFxand dinitrobenzyl-activated glycine, the dinitrobenzyl group providingthe recognition element for this class of Flexizyme. Acylation yieldsranging from 26% to 60% were obtained, as assessed by polyacrylamidegel electrophoresis (PAGE) under acidic conditions .To ask whether RNA aminoacylationwould interfere with or potentiateRNA copying chemistry, we first established a primer extension assayfor RNA copying initiated from a 2′(3′)-aminoacylatedRNA primer 2A. To am26 We diluted the RNA acylation reaction mixture into primerextension buffer, added the template strand and MgCl2 (2.5mM), and initiated primer extension by addition of the C*C dinucleotide(20 mM). Note that due to the short half-life of the ester linkageof 2′(3′)-glycyl RNA under primer extension conditions, and the fact that acylationdoes not proceed to completion, a mixture of 2′(3′)-glycyland native 2′,3′-hydroxyl-terminated RNA was alwayspresent in our Flexizyme-treated reaction mixtures. These two primerspecies are not separated by PAGE employing Tris-borate EDTA (TBE)buffer, although the products of primer extension can be resolved.Upon analysis of the reaction mixture, two new bands were observed,which we hypothesized to be due to +1 extension from either 2′(3′)-glycylRNA or native RNA (vide infra).To investigate the ability of the 2′(3′)-glycyloligonucleotideto act as a primer, we designed a template that provides a singlebinding site for a 5′–5′ aminoimidazolium-bridgedcytidine dinucleotide (C*C), the reactive species in primer extensionusing 2-aminoimidazole-activated cytidine ribonucleotides 2B.26 We nditionsS5E,F, an2 was used in theseassays to allow detection of both +1 bands at earlier time points,as the unmodified RNA primer reacts slowly under these conditions.Reaction mixtures thus treated were identical to those obtained fromcontrol reactions lacking Flexizyme, indicating that the nucleophilicglycyl amino group is required for formation of the novel +1 product.Finally, to directly prove that the free amino group is consumed duringprimer extension, we employed biotin labeling .27 To obtain 2′(3′)-aminoacylated, phosphoramidate-linkedRNA containing only a single-nucleotide extension , we first aminoacylated an RNA primer usingdFx Flexizyme. Following acylation, we performed a primer extensionreaction using a DNA:RNA hybrid template in which the region to becopied is RNA but the remainder of the template is DNA. The templatecontains only a single binding site for the activated C*C imidazolium-bridgeddinucleotide. Incubating the primer template duplex with the C*C dinucleotideleads to robust conversion of the primer to extended products in whichthe +1 nucleotide is connected to the terminal amino group of the2′(3′)-aminoacylated RNA by a phosphoramidate linkage.We omitted Mg2+ from the primer extension reaction to discourageextension of the fraction of the RNA primer that was not acylatedin the Flexizyme reaction. DNase digestion of the template then facilitatedrecovery of the modified primer, which we purified using preparativegel electrophoresis. To obtain RNA strands in which the amino acidlinkage is followed by a longer stretch of ribonucleotides, we replacedthe primer extension reaction with a ligation reaction, using a ligatorRNA oligonucleotide bearing a 2-methylimidazole group activating the5′-phosphate. In this case, the entire template was DNA sothat following ligation, DNase treatment and preparative gel electrophoresisenabled purification of the modified, amino acid-linked strand.To investigate the reactivity of 2′(3′)-aminoacylated,phosphoramidate-linked RNA (“amino acid-bridged RNA”),we required a means of isolating single-stranded RNA containing site-specificphosphoramidate linkages, to study primer extension, ligation, andhydrolysis. We adapted our recently reported strategy for the generationof site-specific 3′–5′ pyrophosphate linkagesto provide a means to access these unusual amino acid-bridged RNAs or an internal 2′(3′)-glycyl phosphoramidatelinkage , using the strategies outlined above. The 2′(3′)-glycyl,phosphoramidate-linked RNAs were then incubated at 22 °C in apH 8.0 solution at a high (100 mM) or low (2.5 mM) concentration of Mg2+, and in the presence or absence of a complementary strand . The maximum stabilitywas observed for duplex products at low concentrations of Mg2+. Under these conditions, we observed half-lives of 53 h for terminallinkages and 72 h for internal linkages. In the presence of 100 mMMg2+, conditions employed for template copying experiments(see below), the stability was decreased, although the observed half-liveson the order of hours are still sufficient to enable amino acid-linkedRNAs to act as templates for further copying cycles (videinfra).Next, we examined the stability of longer amino acid-bridgedRNAsunder conditions relevant to RNA copying chemistry. We first preparedtwo single-stranded RNAs containing either a terminal , consistent with the previouslyreported stability of the phosphoramidate linkage at pH >5.0.9As the gel-based assay does not report on the specificlinkagecleaved (ester vs phosphoramidate), we desalted samples from the cleavageof Figure S5). To quantify thekinetics of glycyl-RNA primer extension, we used a simplified modelof the reaction . At the start of the reaction, due to incompleteacylation by Flexizyme, both aminoacylated and non-acylated primerspecies are present, which cannot be resolved by gel electrophoresis.We monitored the primer extension reaction by PAGE, which can resolvethe two different +1 extended reaction products, with or without abridging amino acid residue. To follow the reaction kinetics, we modeledthe hydrolysis of the aminoacylated primer to the native RNA primeras a first-order irreversible reaction with a rate constant k2. The pseudo-first-order rate constant, k1, for extension of the aminoacylated primerwas estimated by nonlinear regression using independently measuredvalues for the aminoacyl hydrolysis rate constant, k2, the native RNA primer extension rate constant, k3, and the initial fraction of aminoacylatedprimer P0gly .The proportion of amino acid-bridged RNAswithin a prebiotic populationof polynucleotides would depend on the rates of amino acid activationand aminoacylation of RNA, and the competing rates of aminoacyl esterhydrolysis and phosphoramidate linkage formation. To investigate therate of primer extension via phosphoramidate formation, we measuredthe rates of primer extension on a template designed to provide asingle binding site for the C*C imidazolium-bridged dinucleotide (cis-diol or a 2′(3′)-glycylgroup . We note that the rates we reportcombine possible reactions initiated from both 2′- and 3′-linkedglycyl residues. At pH 8.0 and 50 mM Mg2+, the rate ofprimer extension via phosphoramidate bond formation was similar tothat observed for phosphodiester bond formation (k1 = 11 h–1 vs 9 h–1 for RNA). We have previously observed that N–P bond formationusing 2′- or 3′-amino groups is insensitive to Mg2+ concentration.13 This feature is highlydesirable if genetic copying chemistry is to be integrated withinfatty acid vesicles, as concentrations of free Mg2+ of>4 mM degrade and precipitate such membranes. We therefore performedthe same primer extension reactions with and without 2.5 mM Mg2+. The observed rates for primer extension via phosphoramidatebond formation were insensitive to Mg2+, whereas phosphodiesterbond formation was much slower at lower Mg2+ concentrations.In the absence of Mg2+, the rate of primer extension forthe 2′(3′)-glycyl-terminated primer was 2 orders ofmagnitude greater than for extension from the canonical diol-terminatedRNA (k1 = 12.0 h–1 vs0.118 h–1 for RNA). These results are in accordancewith the greater nucleophilicity of the amino substituent relativeto the hydroxyl group and the presumed requirement for divalent metal-mediateddeprotonation of the 3′-hydroxyl to afford the Mg-bound alkoxide,the most likely active species for primer extension of canonical RNA.The much greater reactivity of the glycyl-terminated RNA raised thequestion of whether primer extension from this modified primer isstill dependent on the template. However, we found that primer extensionin the presence of the template yields 76% extended product after15 min compared to only 6% after 30 min in the absence of the template.By following the procedures outlined above, we obtained estimatedrates for primer extension reactions using primers terminated in eithera 2′–3′ 28 and noncanonical nucleotides29 can stallprimer extension, presumably due to the suboptimal geometry of thereaction center. To see if an amino acid bridge would interfere withproper pairing of the terminal primer-template base pair, we quantifiedthe rate of the reaction for a primer in which the terminal 3′-nucleotideis joined by a phosphoramidate linkage to an upstream glycine “bridge”. The observed rate for extensiondownstream of the amino acid bridge was similar to that obtained foran identical primer containing only phosphodiester linkages (10.7h–1 vs 8.8 h–1 for RNA). Thus,the incorporation of a bridging amino acid does not significantlyretard downstream primer extension steps.The incorporation ofmismatched bases16 This scenario is attractive as ligation requiresfewer chemical reaction steps than primer extension to copy a templateof a given length. However, rates of RNA ligation are much lower thanfor polymerization;20 consequently, lossof the activating group competes with ligation, leading to overalllow yields. The slow rate of RNA ligation can be explained by thefact that the leaving group is simply a protonated imidazole, whichlacks the highly preorganized structure of the imidazolium-bridgeddinucleotide intermediate of primer extension. In fact, short oligoribonucleotidesending with 3′-amino-2′,3′-dideoxyribonucleotidesshow ligation rates that are orders of magnitude faster than thoseof all-RNA oligonucleotides.16 However,no potentially prebiotic route to 3′-amino nucleotides is yetknown. We therefore wondered whether the more prebiotically plausible2′(3′)-aminoacylated RNA would show similar trends inrate and yield for non-enzymatic ligation.In addition to thepolymerization of activated nucleotides, RNAtemplates can also be copied by the ligation of short oligomers.Figure S8).To compare the ratesof ligation of unmodified RNA and aminoacylatedRNA, we used a ligation assay similar to that developed for primerextension and the same formalism to model the kinetic system, assumingsaturation of the primer-template duplex with the ligator. For ligationreactions, we were able to determine the rate of formation of theligated product from aminoacylated RNA by collecting data at timepoints at which reaction with the control RNA primer was negligible.As input to our kinetic model, we measured the rates of hydrolysisand the rates of control reactions with an RNA primer in the presenceof the amino acid dinitrobenzyl ester . Although 2-aminoimidazoleis a superior activating group for non-enzymatic polymerization, dueto enhanced formation of the active imidazolium-bridged dinucleotideintermediate, 2-methylimidazole activation is superior for N–Pligation.16 This is consistent with thefact that 2-aminoimidazole is an intrinsically worse leaving group,due to its higher pKa.30 Indeed, in our system, the observed rate of ligation was6-fold greater for the 2MeI-activated ligator (1.81 h–1 vs 0.281 h–1). Notably, the rate of ligation forthe 2′(3′)-glycyl primer reacting with a 2-methylimidazole-activatedligator, at 2.5 mM Mg2+, was ∼500 times greaterthan for the equivalent reaction with unmodified RNA . In the absence of thetemplate, no ligation was observed on the time scale of the experiment.We first tested the template-directed ligation ofa 2′(3′)-glycylprimer with either a 2-methylimidazole- or 2-aminoimidazole-activateddecamer S9. AlthoFigure S8). We tested eight amino acids that differ in charge, size,and stereochemistry. All amino acids, except for Nα-acetyl-l-lysine, reacted orders of magnitudefaster than RNA under the conditions of the assay (pH 8.0 and 2.5mM Mg2+). l-Lys reacted at the greatest rate,which is perhaps surprising given its length and positively chargedside chain. Acetylation of the lysine α-amino group blockedligation completely, while acetylation of the ε-amino groupdecreased the rate 3-fold, confirming regioselectivity for reactionof the α-amine versus the ε-amine . Lysine and aspartate both displayed apreference for reaction of the l-enantiomer, although leucinedisplayed no such preference. Overall, it is notable that amino acidswith different properties could be incorporated into RNA via ligation.For example, the carboxyl and amino side chains introduced by aspartateand lysine, respectively, have no parallel in native RNA. Such integrationof novel functionality may allow for the expansion of the catalyticrepertoire of ribozymes assembled by non-enzymatic ligation.Having demonstrated enhanced ligationrates with aminoacylatedRNA, we were interested in determining whether the ligation rate woulddiffer significantly across a panel of amino acids .To determine whether an RNA strand containing a single amino acidbridge could act as a template for RNA primer extension, we used thebridged 27mer ssRNA 2 (5′-AGAGAA-GAGAGCAGACA-gly-CCCGGCAGCU-3′),and an all-RNA control, as templates. The bridged template containsa stretch of residues 5′-ACA-gly-C-3′ such that onlyone activated dinucleotide intermediate, G*U, was necessary to comparetemplate-directed copying at three positions. We tested three casesthat differed only in the position of the primer 3′-end relativeto the glycine bridge in the template. In the first case, the imidazolium-bridgedG*U dinucleotide spans the glycine bridge 6A. In th2+, which is damaging to protocell membranes at low millimolar concentrations.We have also examined the non-enzymatic, template-directed ligationof an aminoacylated RNA strand to a 2-methylimidizaole-activated ligatorRNA. In this case, rates of ligation are enhanced by at least 2 ordersof magnitude. Similar rate enhancements are seen with primers terminatingin 3′-amino-2′,3′-dideoxyribonucleotides;16 however, no prebiotic synthesis of 3′-aminonucleotides has been described. In contrast, the aminoacylation ofRNA is central to biology.We have found that the aminoacylation of an RNAprimer can, undercertain conditions, greatly enhance the non-enzymatic copying of anRNA template. The reaction of aminoacylated RNA primers with incomingimidazolium-bridged dinucleotides gives RNA products containing anamino acid “bridge” composed of a 5′ ester linkage and a 3′  phosphoramidate linkage.Notably, this copying reaction proceeds in the absence of Mg2 but these reactions are quite inefficient.The discovery of more effective, prebiotically plausible chemistryfor RNA aminoacylation would suggest the potential for a common rolefor this chemistry in the origins of both replication and translation.An efficient chemical aminoacylation process would also be experimentallyuseful if it overcame the sequence limitations enforced by our useof the Flexizyme ribozyme, which acylates only RNA terminating inCA-3′.We employed Flexizyme-catalyzed acylationto obtain high yieldsof aminoacylated RNA primers for our studies. It is possible thatRNA aminoacylation began to play a significant role in the RNA Worldonly after the evolution of ribozymes became widespread, but an earlierprocess would open up the possibility of a role for aminoacylationchemistry in non-enzymatic RNA replication or ligation-mediated ribozymeassembly. The aminoacylation of RNA has been reported from mixturesof phosphorimidazolide-activated nucleotides, imidazole, and aminoacids,The regioselectivity of the phosphoramidate-formingprimer extensionand ligation reactions remains unknown. Our gel-based analysis cannotdistinguish between reactions initiated from the 3′ and 2′esters, because of the rapid transacylation of the initially formedaminoacyl ester. The different regioisomers, if a mixture indeed resultsfrom phosphoramidate formation, may display different templating activitiesand stabilities that have been conflated in this study.9 Ourstability studies revealed the protective effect of duplex formation,which enhances the kinetic stability of amino acid-bridged RNA approximately2-fold. Notably, high concentrations of Mg2+ promote degradationof the amino acid “bridge”; taken together with themuch enhanced rates of RNA copying observed at low concentrationsof Mg2+, this result suggests that amino acid-bridged RNAwould accumulate preferentially under low-free Mg2+ conditions,conditions that are also most favorable for protocell stability. Themajor products of degradation of an amino acid “bridge”under RNA copying conditions are a 5′-fragment composed ofnative RNA, resulting from aminoacyl ester cleavage, and a 3′-fragmentbearing an amino acid at the 5′-terminus linked by a phosphoramidatelinkage. Such 5′-N-linked amino acids have been shown to behighly competent for further extension into peptides under activatingconditions.31 Thus, phosphoramidate bondformation via either non-enzymatic primer extension or ligation, followedby hydrolysis of the aminoacyl ester, could initiate peptide synthesis,in addition to the functions outlined above.It hasbeen noted previously that the enhanced lifetime of thelinkage of the aminoacyl ester to RNA upon formation of a neighboringphosphoramidate linkage may provide a mechanism for the stable integrationof amino acid functionality into RNA.32 In addition, introducing novel functional groupsto RNA via chemical modification has proven to be a powerful approachfor obtaining ribozymes with enhanced33 or new-to-nature functions.34 Our resultsshow that non-enzymatic ligation with different amino acids can furnishRNA strands with bridging amino acids with a range of side chains.This novel route to the integration of amino acids within RNA mayprovide new opportunities for ribozyme catalysis that would be excitingto test.We have shown thatRNA containing an amino acid bridge remainscompetent as a template for further cycles of copying. It remainsunknown whether amino acid-bridged RNA may serve a catalytic function.Ribozyme function can be enhanced using free amino acids as cofactors.


Met sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.Herein, we present a multi‐cycle chemoenzymatic synthesis of modified RNA with simplified solid‐phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid‐phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light‐controllable RNA nucleotides into the tRNA Synthetic access to modified RNA is of high importance. Both the gold standard—chemical solid‐phase synthesis—and enzymatic methods have limitations. The present study offers a best‐of‐both‐worlds approach and enabled the synthesis of a tRNA derivative with four modifications. This approach is compatible with a whole toolbox of light‐reactive and other modifications. RNA plays an essential role in many biological processes. In order to understand the underlying intricate mechanisms and to learn about the structural and dynamic aspects especially of regulatory RNA, there is increasing demand for reliable methods to synthesize modified RNA with novel functions, different stability and a variety of biophysical probes.The traditional automated chemical solid‐phase synthesisT7 RNA polymerases WLight‐responsive modifications find numerous applications in oligonucleotide research.28, 29T4 RNA ligase 1 (Rnl1).T4 RNA ligase 2 (Rnl2) and a subsequent digestion of the DNA splint with DNase.Our chemoenzymatic method consists of three enzymatic steps. In the first step, an existing RNA is 3′‐extended with a nucleoside 5′,3′‐bisphosphate using In this study, we establish the repeated application of this procedure Scheme  and applNPEppC, NPEppG and NPEppU or the bisphosphate mAzo)pp) was used.RNA1–RNA5) with lengths that can be readily prepared using solid‐phase synthesis and purified by HPLC.For the modification, a set of nucleotide bisphosphates (RNA1 (orange sequence in Scheme NPEppC or mAzo)pp as well as a reduction in reaction time (from an overnight reaction to 3–8 h). For comparison, the reaction was also performed in solution .n Figure  with no However, the significant handling advantage of the solid‐phase‐supported procedure was apparent, because in solution the workup of every step required either precipitation or RP‐HPLC, while the solid‐phase route required only washing. Importantly, if a purification step was required after one of the operations on solid support or if the beads degraded during the repeated operations, our particular choice of immobilization technique would allow releasing the sequence from the beads, carrying out a purification step and reattaching the sequence to new beads to continue the synthesis.NPE‐RNA2RNA1‐C) was 3′‐extended with either NPEppU or mAzo)pp of up to 17 % and 22 % for NPE‐RNA2‐UNPERNA1‐C and mAzo‐RNA2‐mAzoRNA1‐, respectively pppppppp but rather by the combination of both domains, where each of them is strong, that the best results are obtained.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Porcupine quill injuries are common in dogs; however, intracardiac quill migration is rare. Considering the high rate of complications secondary to quill penetration and migration, veterinarians should be aware of risks associated with long-standing foreign bodies even when no clinical signs are evident. A dog was referred for three episodes of recurrent fever that resolved with antibiotic therapy. Ultrasonography and computed tomography revealed a single linear hyperechoic structure traversing the interventricular septum from the heart base to the cardiac apex. A median sternotomy was performed, and a porcupine quill was removed under a direct ultrasound guide. The dog recovered uneventfully. Dogs without evidence of severe cardiac injury secondary to a single intracardiac quill may have a good prognosis.Hystrix cristata, (HC)] migration through the heart in a dog and the use of multimodality imaging for the diagnosis and surgical approach. A 4-year-old 25 kg female mixed-breed dog was presented for evaluation of recurrent fever. Transthoracic echocardiographic examination showed a linear, hyperechoic foreign body traversing the interventricular septum from the heart base to the cardiac apex. Computed tomography and transesophageal echocardiography confirmed the presence of a single structure resembling a quill; a later esophageal endoscopy showed no anatomical alterations or mucosal injuries. Following median sternotomy and after accurate localization by intraoperative ultrasound, the quill was successfully removed. The dog had a good recovery without evidence of severe cardiac injury secondary to an intracardiac quill. To the best of the authors’ knowledge, the identification of an intracardiac HC quill by ultrasonography and CT and its successful removal by ultrasound guide has not been previously reported.The porcupine is a nocturnal quill-bearing rodent common throughout Italy. When threatened, it defends itself by erecting its quills, rattling its tail, and running sideways or backyard into predators. The quills are released upon contact with assailants and tend to migrate into several body tissues or cavities due to the unique inverted cuticles (crenate) directed downwards to the quill base (a sort of “harpoon effect”). Intracardiac migration of porcupine quills has been reported in a few dogs with severe clinical signs. This case report describes a single porcupine quill [ Hystrix cristata) is a quite common rodent that lives in Italy, mainly in wooded and bushy areas. The antipredator behavior of these rodents is quite fascinating: tail rattling, in most cases, is sufficient to move away solitary predators, while backyard/sideways attacks are exhibited only in extreme situations of direct strikes [HC quills have a cylindrical shape in the lower two-thirds of the shaft and then flatten toward the apex; they can reach a significant length of 380–400 mm, while the thickness is about 2 mm. Moreover, there are up to 12 furrows on the shaft, and they correspond to the septa dividing the thick alveolar medulla [Erethizon dorsatum), are significantly shorter (85 mm), smooth, weakly bent, and slightly flattened; the tip is barbed (microscopic backward-facing deployable barbs), and the alveolar medulla is strongly developed but is not divided by thick septae [Hystrix spp. Is sharp and not barbed, whereas the external end has a unique inverted cuticle directed downwards to the quill base (like a “harpoon”) [Erethizon dorsatum) reduce the possibility of pulling out the quills and aids penetration and migration through the tissues [The European porcupine , a mild hyporegenerative anemia  and elevated alkaline phosphatase .A 4-year-old female, 25 kg, mixed breed, was presented to the Veterinary Teaching Hospital of the University of Perugia for evaluation of recurrent fever. Four months prior to the presentation, the dog had been evaluated by the referring veterinarian for swelling and pain in the ventral region of the neck associated with a presumptive snake bite and had been treated with antibiotic/corticosteroid therapy. Since this event, the dog has presented three episodes of recurrent fever resolved with antibiotic therapy. No other clinical signs were reported by the owner and referring veterinarian. At the time of presentation to our Hospital, the dog showed lethargy, fever (40.4 °C), and mild tachypnoea (28 breaths/min). A complete blood count and serum chemistry were performed. Abnormalities included a neutrophilic leukocytosis  and a linear hyperechoic structure traversing the interventricular septum from the heart base to the cardiac apex was visualized. In the longitudinal plane, the foreign body appeared as two well-defined mural parallel hyperechoic lines, with multiple additional hyperechoic lines parallel to the walls , lactated Ringer’s solution (2 mL/kg/h IV), and meloxicam . For pain management, the initial treatment included methadone  0.2 mg/kg IM q4 h. Two days post-operatively, results from the cultures revealed the growth of an amoxicillin-resistant Pseudomonas aeruginosa with sensitivity to amikacin, gentamicin, enrofloxacin, and marbofloxacin. Therefore, antibiotic therapy was changed to enrofloxacin . A few days after the surgery, a surgical site infection (SSI) of the wound involving pectoral muscles was noted and treated by surgical curettage of the sternal area, local swab for antimicrobial testing, and abundant lavage. Two days later, preliminary culture results from the infected wound indicated growth of Pseudomonas aeruginosa that was only sensitive to clindamycin, amikacin, gentamicin, enrofloxacin, and marbofloxacin. Amikacin  was infiltrated locally three times a week. The wound improved, and the draining tract resolved, so the dog was discharged after four weeks after the surgery.After formal consent of the owner, the patient underwent general anesthesia, using a standard clinical protocol: fasting for 12 h and free access to water; premedication was performed with methadone ; 10 min after premedication, anesthesia was induced with propofol  and midazolam  through a cephalic venous cannula previously and aseptically placed. Following tracheal intubation, the dog was connected to a rebreathing circuit (50 mL/kg oxygen flow) and maintained with isoflurane  in 100% oxygen. The patient was mechanically ventilated during anesthesia. Lactated Ringer solution (10 mL/kg/h CRI) was infused during the entire anesthetic procedure. Meloxicam  and cefazolin  were administered during patient preparation and at least 120 min prior to surgery. After the routine aseptically preparation of the operative field, a ventral midline sternotomy was performed. Intraoperative echocardiography was used to evaluate the localization of the foreign body and confirm its position through the interventricular septum. The pericardium did not show any modification. A small pericardiectomy (4 × 4 cm) was carried out to provide a clearer exposure of the epicardium wall. A small bulging round area was noted at the apex of the left ventricular wall; however, the quill was not yet visible on the cardiac surface. An ultrasound probe, encased in a sterile protective cover, was placed over the cardiac apex and intraoperative ultrasonography allowed to visualize the quill and its tip. A double horizontal mattress suture tension (3-0 polypropylene) was placed in the epicardium wall around the area of the tip of the quill (at about 10 mm from the apex of the heart). Under ultrasonographic direct visualization, a small (8 mm) incision of the ventricular wall was carried out by n. 11 scalpel blade; the depth of the incision was about 6 mm; through the incision, a small, curved tip hemostatic forceps were introduced, and under ultrasound guide, the quill was grasped and gently pulled out from the heart . No cardFollow-up six months after the surgery revealed that the dog was working to full capacity without evidence of any clinical signs.HC quill and its surgical removal using the intraoperative ultrasonographic guide.To the authors’ knowledge, this is the first clinical case reporting an ultrasound and CT visualization of an intramyocardial Porcupine quills are unique among migrating foreign bodies, and their injuries involve more areas of the body. Most commonly, the areas involved are represented by the head and neck, followed by limbs and trunk in dogs . In caseIn the veterinary literature, migration of foreign bodies such as grass awn, sewing needles, wooden skewers, metallic projectile, and catheter fragments into the heart has been previously reported ,22,23,24Currently, there are no indications regarding the management of a long myocardial quill in veterinary medicine. Surgical intervention has been debated for small foreign bodies, such as quills, sewing needles, projectiles, and skewers, in humans and animals ,22,25,26sternebrae to the xiphoideus process was preferred permitted the use of ultrasound for identifying the quill and its tip. Indeed, it would be advisable to grasp the tip of the foreign body and not the tail (because of the inverted cuticle). Moreover, this approach allowed us to pull out the quill with the correct line of extraction since the thorn was positioned along both septa following the longitudinal axial of the heart. Left lateral access would not have had the same easy access to retrieve the quill in a straight line because of the presence of the caudal rib. Moreover, the safe removal of the thorn from the heart goes through a correct identification of the quill by ultrasound probe in order to evaluate the direction of the thorn and to determine the correct maneuver for its removal. Since foreign bodies like quills become less rigid and more friable the longer they remain in tissues making them more susceptible to breakage during removal [Thoracoscopic exploration and removal of porcupine quills have been previously described in humans; however, postoperatively, complications due to persistent porcupine quill migration were observed . In our  removal , the manIn our clinical case, the quill was localized into the interventricular septum; in a different scenario, where a quill perforates the atrial or ventricular wall and/or damages the valves, an attempt to remove the foreign body, without performing a cardiopulmonary bypass could lead to catastrophic consequences due to hemorrhage across the septum or systemic air embolism . In our In our specific case, doubt arose about the fact that retrieval of the quill could be challenging since its embedding into the myocardium. Furthermore, the closure of the myocardium could generate some technical difficulties. Thankfully, the retrieval was smooth and easy. The pulling-out effort was minimal, and no embedding issues were experienced. Ultrasound re-checking after removal did not point out any small or fragmented parts of the extracted quill that may become embedded in deeper tissues of the heart. Closure of the epicardium was easy and fast using the same tension sutures previously applied.Interestingly, despite the fact that the quill migrated through the thoracic cavity, no air leakage or scars were identified from any of the lung lobes during the direct inspection, suggesting that the quill did not perforate any lung lobes.Pseudomonas aeruginosa similar to what was revealed from the quill and his housing into the heart. We hypothesize that a passage of infection through the sternum and pectoral muscles occurred from the quill housing into the heart, creating SSI of the wound. P. aeruginosa is an opportunistic pathogen that causes a wide spectrum of infections and leads to substantial morbidity in immunocompromised patients. Frequently, P. aeruginosa demonstrates resistance to multiple antibiotics, as in our case, jeopardizing the selection of appropriate treatment. In the dog described in our case, the long therapy with enrofloxacin and amikacin locally infiltrated [As mentioned before, a few days after recovery, the dog showed SSI of the caudal part of the wound in the area of the pectoralis muscles. Results of bacteriological culture showed iltrated  significPorcupine quill injuries are common in dogs, although intracardiac migration is rare and is commonly associated with severe clinical signs. Diagnosis of intracardiac porcupine quill migration in dogs without evidence of severe cardiac injury can be a challenge for clinicians. An accurate diagnosis and identification of the quill by a multimodality imaging approach is mandatory for planning a safe removal from the myocardium. Moreover, intraoperative ultrasound is useful in guiding the extraction of the foreign body.


While coral reefs in Australia have historically been a showcase of conventional management informed by research, recent declines in coral cover have triggered efforts to innovate and integrate intervention and restoration actions into management frameworks. Here we outline the multi-faceted intervention approaches that have developed in Australia since 2017, from newly implemented in-water programs, research to enhance coral resilience and investigations into socio-economic perspectives on restoration goals. We describe in-water projects using coral gardening, substrate stabilisation, coral repositioning, macro-algae removal, and larval-based restoration techniques. Three areas of research focus are also presented to illustrate the breadth of Australian research on coral restoration, (1) the transdisciplinary Reef Restoration and Adaptation Program (RRAP), one of the world’s largest research and development programs focused on coral reefs, (2) interventions to enhance coral performance under climate change, and (3) research into socio-cultural perspectives. Together, these projects and the recent research focus reflect an increasing urgency for action to confront the coral reef crisis, develop new and additional tools to manage coral reefs, and the consequent increase in funding opportunities and management appetite for implementation. The rapid progress in trialling and deploying coral restoration in Australia builds on decades of overseas experience, and advances in research and development are showing positive signs that coral restoration can be a valuable tool to improve resilience at local scales . RRAP is focused on creating interventions to help coral reefs at multiple scales, from micro scales  to large scales  to resist, adapt to and recover from the impacts of climate change. None of these interventions aim to single-handedly restore the entirety of the Great Barrier Reef, nor do they negate the importance of urgent climate change mitigation action. Historically, management of coral reefs in Australia has focused on reducing local and regional stressors. These include, for example, overfishing (mainly through zoning and enforcement), mining for oil and gas exploration, coastal development , and poor water quality (through land management and improved technology). Apart from targeted crown-of-thorns starfish (COTs) control, which has since 2013 become an important part of reef management on the Great Barrier Reef (GBR) ), coral Porites spp., away from a harbour development site to avoid physical damage from dredging activities -1) and cWith the promising outcomes of larval-based restoration trials on the GBR, a pilot capacity building project called Boats4Corals investigated whether boats and crews from the reef tourism industry and Indigenous Traditional Owners in the Whitsundays region could undertake larval restoration operations using modified larval culture pools on reefs , 46, ini2) onto devices for deployment, aims to assist reef recovery and restoration by enhancing the low natural survival characteristic of post-settlement and post-deployment phases  the ability to manipulate the benthic community surrounding the coral to control settlement inducers and competitors, (iii) the opportunity to overcome challenges associated with natural settlement processes on degraded reefs, including a lack of appropriate settlement cues or available substrate [Coral seeding, defined as the settlement of coral larvae or the attachment of small fragments or micro-fragments (<1 cmiewed in ). While ubstrate –55 and tubstrate –58, and ubstrate –63. Coraubstrate  or speciubstrate  and 3D pubstrate . More reubstrate . BuildinAcropora tenuis spat survival three-fold over featureless devices [Acropora millepora, Acropora muricata and Montipora aequituberculata were deployed across three sites in the inshore Keppel Islands in January 2021 (~ six week old spat). Surveys of devices in April 2021 indicated early survival yields averaging 50% on plugs and >95% on devices, and differences in survival amongst sites. Ongoing surveys of devices are planned for throughout 2021, and aim to identify the drivers of site and species-level differences in survival and growth to allow future optimisation of coral seeding restoration efforts. Taken together, these projects demonstrate the capacity to improve the survival of deployed spat and coral fragments above natural rates and suggest that seeding devices may offer an additional tool that, when combined with other techniques and advances in automation and high throughput technology, may offer a valuable tool for the reef restoration toolbox.Recent research, funded by the Australian Institute of Marine Science (AIMS), has identified some functional features, such as wide grooves in the settlement surface, that improved  devices , 63. A s devices . BuildinThe Reef Restoration and Adaptation Program (RRAP), was initially funded by the Australian Government to scope existing and novel technologies that could help support the resilience of the GBR, and to deliver an investment case for further research and development of reef interventions , 67. ThiThe RRAP feasibility study found there was no single solution to supporting the Reef’s resilience , 68. RatThe targeted research and development program will balance minimising risk with maximising the opportunity to save coral species and values to create a suite of interventions that would take an integrated, three-pronged approach: 1) cooling and shading the reef to help protect it from the impacts of climate change 2) assisting reef species to adapt to the changing environment, and 3) supporting the recovery of degraded reefs through restoration; over seven intervention research and development subprograms . The focThe envisaged final phase of RRAP would be an implementation phase which could overlap with the research and development phase as early as five years from commencement, depending on the specific intervention. This would include commercial transfer of the reduced set of candidate intervention measures, including the construction, operation, and deployment of full-scale systems.The current and projected rates of climate warming combined with the devastating effects of heat-induced coral bleaching on reef health indicate that corals need to rapidly adapt to become more thermally tolerant if they are to survive into the future. Assisted evolution is a collective term for several interventions aimed at improving coral performance through the acceleration of naturally occurring evolutionary processes . To dateThere are three main approaches to assisted evolution:Managed or selective breeding refers to approaches based on sexual reproduction, such as cross-breeding between distinct populations of the same species  or between species , and the cross-breeding of comparatively heat tolerant colonies from the same population [pulation . These apulation –77 especpulation .(Pre)conditioning is the exposure of organisms to sublethal stress which may elicit an increased tolerance to subsequent stress exposures. Prior exposure to elevated temperatures can reduce bleaching susceptibility in adults  and and(Pre)s . Ho. Ho(Pre)Microbiome manipulation or microbiome engineering is the manipulation of individual microbes, microbial communities, or their hosts . Promisgnition, . The devgnition, , but thignition, –91.A less often discussed assisted-evolution approach is the managed formation of coral chimeras, for instance through the use of microfragments of full siblings to reskin dead skeletons of large, massive colonies . Close rCurrently, assisted evolution for corals is in a research and development phase and is not yet implemented on the GBR. The feasibility of assisted evolution has mostly been tested under laboratory conditions and in small field trials (Acropora impacted by recent heat waves) can exist along broad latitudinal gradients stretching >2000km, exposing species to >7°C temperature difference. Such broadly distributed coral species typically exhibit different thermal optima tuned to local conditions  that apns (e.g. . Reefs ins (e.g. ). Whilstns e.g. , 103), “, “Acropons  mangrove lagoons, are exposed to variable environmental conditions including changes in pH , extremes in temperature, and amplified metabolic activity , 109. Thgenation , yet appgenation ). These genation , 111).Together these various examples of naturally heat tolerant corals demonstrate existing natural variation in thermal tolerance and therefore provide important genetic reservoirs to support reef restoration and adaptation activities. However, key knowledge gaps remain. For example, the impacts of selective breeding on genetic diversity and local adaptation, or the capacity for corals to maintain traits among generations still warrant further studies. Reciprocal transplant and gardening experiments of populations and genetic crosses of corals are now underway to understand genetic and environmental drivers of coral fitness among reefs . UltimaRecent studies clearly describe how the impacts of climate change, such as major bleaching events, are keenly felt by reef users and visitors. They report diminished experience quality, less confidence that their own actions will matter, and emotional responses consistent with ‘ecological grief’ . These iWithin Australia, as elsewhere, there are programs of community action and partnerships between citizens and researchers that encourage participation in monitoring of coral health and in coral restoration activities. These efforts often intersect with long-standing marine citizen-science programs that are sponsored by non-governmental organisations, education and research providers or agencies with responsibility for marine park management. This can contribute to environmental literacy and citizenship amongst participants . FurtherWhen approaching politically and socially contested proposals such as large-scale restoration and adaptation of the GBR, there are a number of complex social and institutional considerations. The first of these is understanding the social acceptability (or otherwise) of proposed restoration interventions amongst the broader community, key rights-holders such as Indigenous Traditional Owners, stakeholders such as reef-using or reef-dependent economic sectors, and other engaged interest groups. The second challenge is assessing how proposed interventions (or non-intervention) are likely to affect the diverse social and cultural values, uses and benefits for those groups that associate with the Reef. Third, there is a need to develop appropriate and meaningful ways to engage different groups and interests in the design, deployment and evaluation of proposed interventions or technologies over time. A recent review identified several distinct types of challenges researchers face when working on assisted regional ecosystem adaption including scientific conflicts and debates over the “facts”; social and governance challenges; epistemic challenges, and ontological conflicts . AddressThere are normative reasons  and instrumental reasons  why understanding social acceptance, assessing likely impacts on diverse values, and designing appropriate engagement processes are important. They are normatively important because there are democratic, moral and ethical obligations to involve people in decisions and actions that may affect their livelihoods or future well-being. They are also instrumentally important in that good participation or co-design can lead to enhanced performance of the planned environmental intervention through better tailoring to local conditions, the use of local and traditional knowledge and reduced stakeholder conflict . CriticaSince 2017, there have been approximately 19 in-water coral reef restoration projects, and a rapidly growing field of research into coral restoration and adaptation on the GBR including RRAP. This reflects an increasing urgency for action to confront the coral reef crisis, and an increase in funding opportunities and management appetite for the implementation of interventions. Generally, there has been a collaborative, positive spirit, and coral restoration has brought together stakeholders who rarely work together . However2 of coral reefs [Australia has about 50,000 kmal reefs  and no eal reefs . It is nal reefs . Coral rAdditionally, there is also value in small-scale interventions, particularly at high-value sites where objectives of restoration align with economic and/or cultural incentives through increasing community engagement or the amenity value of tourism hotspots. Since 2018, at least ten tourism organisations have been actively involved in coral restoration projects on the GBR, motivated by the desire to be proactive in improving the condition of the reefs they use . While tThe novelty of the many intervention strategies presented here represent an important shift in the use of coral restoration—away from assessing the feasibility and efficacy of individual techniques, towards developing a holistic approach that includes coordinated and practical solutions with specific objectives linked to long-term outcomes in a changing climate. In Australia, this shift is greatly facilitated by large investments through the RRAP Program. There has also been a shift from research and development to implementation. This approach requires research partnerships that span beyond the coral research space to include Traditional Owners, engineers, social scientists, modellers, economists, infrastructure development experts, and project managers.The rapid progress in trialling coral restoration methods in Australia builds on decades of overseas experience. Australia is now investing considerable time and money into developing novel interventions and consequently, is fast becoming an emerging leader in this space. There are great advantages to continuing to actively collaborate with researchers internationally and with other sectors not currently involved in coral reef management. This could be facilitated through coordinating organisations and networks such as the Coral Restoration Consortium, the International Coral Reef Initiative, RRAP, the United Nations, and the International Coral Reef Society to hasten the optimisation of reef restoration interventions and their contribution to future increases in reef resilience.The challenge for the future include: (1) demonstrating that active restoration can play a meaningful role in improving reef condition through supporting resilience and increasing adaptation when other, less interventionist, management strategies have not worked, (2) implementing these interventions at larger scales, and (3) understanding and mitigating any potential risks from active restoration. At any scale, coral restoration and adaptation interventions do not take away the need for urgent reductions in greenhouse gas emissions.S1 Appendix(DOCX)Click here for additional data file.


The biomimetic fabrication of 3D biology models confers design freedom across various applications. Taking inspiration from nature and using nature as a toolbox have helped in the production of effective and versatile tools that are integrated with modern technology principles and, importantly, perform quite similarly to natural living systems. Exploiting nature’s design principles can contribute to the improvement of the understanding and development of creative products in both fundamental research and industrial applications . To addrBungon et al. developed a promising GFET biosensor for sensing and targeting clusterin, a prominent protein biomarker of Alzheimer’s disease. Considering the remarkable properties of graphene such as its high carrier mobility, current carrying capacity, thermal conductivity, optical properties, and mechanical stability, the material has been employed in FET biosensing platforms. The quantitative responses of the GFET sensors were determined using direct current 4-probe electrical resistance measurements, which demonstrated a limit of detection for the biosensors of four fM.Alzheimer’s disease is the most common degenerative disorder in the world. The combination of naturally occurring minerals in nature such as graphene and capturing biomolecules such as antibodies offers a new opportunity for developing simple diagnostic devices with remarkable performance. The challenging task in developing a biosensor is building a sensing matrix that allows the sensitive and selective capture of biomarkers with low limits of detection. Damiati and Damiati developed a strategy based on machine learning using artificial neural networks (ANNs) as an in silico tool, a 3D flow-focusing microfluidic chip as a simple device to produce MPs, and poly  (PLGA) as a matrix material for the encapsulation of indomethacin (IND), a type of NSAID. The developed strategy represents a powerful tool for predicting and generating size-tunable PLGA MPs. The ability to control the particle size and particle-size distribution is critical in the pharmaceutical industry. The generation of small sizes and narrow size distributions is usually favorable for obtaining final particles with high stability and long shelf-lives, as well as enhancing the bioavailability of poorly soluble drugs.Drug development is a time consuming and expensive process. Many factors may delay drugs’ approval and their availability on the market. For example, the limited utilization of several non-steroidal anti-inflammatory drugs (NSAIDs) is attributed to their poor water solubility. Hence, controlled-release carriers to improve solubility and increase bioavailability are highly demanded. The encapsulation of NSAIDs within a polymeric matrix would allow the sustained and controlled release of the loaded drugs. Furthermore, with the continuous development of artificial intelligence (AI) technologies, the frontiers for polymeric particles and drug-delivery carriers have been greatly advanced. To speed up a drug candidate’s progress, combining machine learning and microfluidic technologies, alongside using biodegradable polymers, represents a promising means for generating drug-loaded polymeric microparticles (MPs). ex vivo and in vivo bone regeneration. Authors review the possibility of seeding mammalian cells and synthetic RNA molecules onto natural or synthetic scaffolds such as polymers and metals to fabricate osteogenic implants. The advantages and limitations of the RNA-based scaffolds are also discussed. Authors highlight using the CRISPR-based genome editing technology to guide RNA scaffolds in bone regeneration. CRISPR/Cas9 represents a promising tool for elucidating various mechanisms of osteogenesis and bone healing in a cost- and time-efficient manner, as well as providing a new strategy for controlling bone infection.Mimicking natural bone for bone grafting has been achieved by taking advantage of the cell’s molecular machinery. Many researchers focus on the development of gene therapy and understanding the mechanisms of bone remodeling to create functional bone tissue that can cure bone defects. In this regard, Damiati and El-Messeiry review combinations of cells, biomaterial scaffolds, and bioactive molecules such as RNA coatings. Different types of RNA including messenger RNA (mRNA), RNA interference (RNAi) molecules, small interfering RNA (siRNA), and long non-coding RNA (lncRNA) play roles in cellular Due to expectations that the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causing coronavirus infectious disease (COVID-19), will continue to spread and might become a common seasonal infection, there is global interest in fabricating filters that can remove the virus from the air. As part of ongoing efforts to find several techniques that can be applied to the bio-decontamination of air, El-Tanbouly et al. review the utilization of indoor plants as an eco-friendly tool to remove air pollutants and habituated airborne microbes. In comparison to filtration and radiation, which are expensive and not feasible for home use, indoor plants are a cost-efficient means of indoor air purification that can be easily adapted to an environment, without special requirements. Phytoremediation technologies commonly use plants to remove hazardous contaminants from environments. Besides the fact that indoor plants are natural air filters that purify air through different mechanisms involving absorption, filtration, dilution, and precipitation, plants have the ability to release small amounts of secondary metabolites and their derivatives, such as polyphenols and alkaloids, into the air. The released compounds show antimicrobial actions and can efficiently interact with the surrounding environment, which may contain airborne microbes. Moreover, regulating humidity in the indoor environment using indoor plants may be useful for reducing SARS-CoV-2 transmission and, thus, in fighting COVID-19.In summary, the collection of articles in this Research Topic highlights the diverse applications of synthetic biology. These studies offer valuable insights to nature inspired technology and will inspire scientists to extend the future of biomimetic products and to deploy biomimetics in various fields.