Ligation Activity Research Articles

Improved one-pot protein synthesis enabled by a more precise assessment of peptide arylthioester reactivity

By investigating 17 peptide arylthioesters that were previously challenging to produce, this study reveals a clear correlation between increased ligation activity and decreased pKa values of their corresponding arylthiols. The observed differences are attributed to variations in thioester bond strength and steric hindrance. These insights have led to the development of an improved one-pot chemical protein synthesis approach that leverages the reactivity differences between peptide arylthioesters with C-terminal Ala-S-Ph(4-NO2) and Ala-S-Ph(2,6-diCH3). This approach eliminates the need for thiol-thioester exchange and additive removal steps while enabling in situ desulfurization, thereby significantly simplifying the protein synthesis process.

Teal-light absorbing cyanobacterial phytochrome superfamily provides insights into the diverse mechanisms of spectral tuning and facilitates the engineering of photoreceptors for optogenetic tools

Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.

Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.

During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.

R2D ligase: Unveiling a novel DNA ligase with surprising DNA-to-RNA ligation activity.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO4 2- end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.

Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

An unexpected stereochemical effect of thio-substituted Asp in native chemical ligation

This study presents an unexpected finding that the cis isomer of β-thio-Asp exhibits higher ligation activity than the trans isomer. This discovery sheds light on the intricate nature of native chemical ligation and highlights the importance of factors beyond the steric effects of the side chain in modulating ligation activity.

Horizontal acquisition of a DNA ligase improves DNA damage tolerance in eukaryotes

Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In this study, bdelloid rotifer Adineta vaga is used as a model to decipher the molecular basis of their extreme tolerance. Proteomic analysis shows that a specific DNA ligase, different from those usually involved in DNA repair in eukaryotes, is strongly over-represented upon ionizing radiation. A phylogenetic analysis reveals its orthology to prokaryotic DNA ligase E, and its horizontal acquisition by bdelloid rotifers and plausibly other eukaryotes. The fungus Mortierella verticillata, having a single copy of this DNA Ligase E homolog, also exhibits an increased radiation tolerance with an over-expression of this DNA ligase E following X-ray exposure. We also provide evidence that A. vaga ligase E is a major contributor of DNA breaks ligation activity, which is a common step of all important DNA repair pathways. Consistently, its heterologous expression in human cell lines significantly improves their radio-tolerance. Overall, this study highlights the potential of horizontal gene transfers in eukaryotes, and their contribution to the adaptation to extreme conditions.

Structural and functional studies of PCNA from African swine fever virus.

Proliferating cell nuclear antigen (PCNA) belongs to the DNA sliding clamp family. Via interacting with various partner proteins, PCNA plays critical roles in DNA replication, DNA repair, chromatin assembly, epigenetic inheritance, chromatin remodeling, and many other fundamental biological processes. Although PCNA and PCNA-interacting partner networks are conserved across species, PCNA of a given species is rarely functional in heterologous systems, emphasizing the importance of more representative PCNA studies. Here, we report two crystal structures of PCNA from African swine fever virus (ASFV), which is the only member of the Asfarviridae family. Compared to the eukaryotic and archaeal PCNAs and the sliding clamp structural homologs from other viruses, AsfvPCNA possesses unique sequences and/or conformations at several regions, such as the J-loop, interdomain-connecting loop (IDCL), P-loop, and C-tail, which are involved in partner recognition or modification of sliding clamps. In addition to double-stranded DNA binding, we also demonstrate that AsfvPCNA can modestly enhance the ligation activity of the AsfvLIG protein. The unique structural features of AsfvPCNA can serve as a potential target for the development of ASFV-specific inhibitors and help combat the deadly virus. IMPORTANCE Two high-resolution crystal structures of African swine fever virus proliferating cell nuclear antigen (AsfvPCNA) are presented here. Structural comparison revealed that AsfvPCNA is unique at several regions, such as the J-loop, the interdomain-connecting loop linker, and the P-loop, which may play important roles in ASFV-specific partner selection of AsfvPCNA. Unlike eukaryotic and archaeal PCNAs, AsfvPCNA possesses high double-stranded DNA-binding affinity. Besides DNA binding, AsfvPCNA can also modestly enhance the ligation activity of the AsfvLIG protein, which is essential for the replication and repair of ASFV genome. The unique structural features make AsfvPCNA a potential target for drug development, which will help combat the deadly virus.

Molecular Anatomy of the Class I Ligase Ribozyme for Elucidation of the Activity-Generating Unit.

The class I ligase ribozyme consists of 121 nucleotides and shows a high catalytic rate comparable to that found in natural proteinaceous polymerases. In this study, we aimed to identify the smaller active unit of the class I ligase ribozyme comprising ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Based on the three-dimensional structure of the class I ligase ribozyme, mutants were prepared and their ligation activities were analyzed. Sufficient ligation activity was maintained even when shortening to 94 nucleotides. However, because it would be difficult to approach the target of ~50 nucleotides by removing only the partial structure, the class I ligase ribozyme was then split into two molecules. The ligation activity was maintained even when splitting into two molecules of 55 and 39 nucleotides. Using a system with similar split ribozymes, we analyzed the ligation activity of mutants C30, C47, and A71, which have been previously identified as the positions that contribute to catalytic activity, and discussed the structural basis of the activity of these bases. Our findings suggest the rationale for the class I ligase ribozyme's assembling from multiple fragments that would be achievable with prebiotic synthesis.

Boronic Acid Assisted Self-Assembly of Functional RNAs.

Boronate esters formed by reaction of an oligonucleotide carrying a 5'-boronic acid moiety with the 3'-terminal cis-diol of another have been shown previously to assist assembly of fragmented DNAzymes. Here we demonstrate that boronate esters replacing the natural phosphodiester linkage at selected sites of two functional RNAs, the hairpin ribozyme and the Mango aptamer, allow assembly of functional structures. The hairpin ribozyme, a small naturally occurring RNA that supports the reversible cleavage of appropriate RNA substrates, is very sensitive to fragmentation. Splitting the ribozyme at four different sites led to a significant decrease or even loss of cleavage and ligation activity. Ribozymes assembled from fragments capable of boronate ester formation showed restoration of cleavage activity in some but not all cases, dependent on the split site. Ligation proved to be more challenging, no supportive effect of the boronate ester was observed. Split variants of the Mango aptamer also showed a dramatic loss of functionality, which however, was restored when 5'-boronic acid modified fragments were used for assembly. These studies show for the first time that boronate esters as internucleoside linkages can act as surrogates of natural phosphodiesters in functional RNA molecules.

Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications.

Untranslatable mitochondrial transcripts in kinetoplastids are decrypted post-transcriptionally through an RNA editing process that entails uridine insertion/deletion. This unique stepwise process is mediated by the editosome, a multiprotein complex that is a validated drug target of considerable interest in addressing the unmet medical needs for kinetoplastid diseases. With that objective, several in vitro RNA editing assays have been developed, albeit with limited success in discovering potent inhibitors. This manuscript describes the development of three hammerhead ribozyme (HHR) FRET reporter-based RNA editing assays for precleaved deletion, insertion, and ligation assays that bypass the rate-limiting endonucleolytic cleavage step, providing information on U-deletion, U-insertion, and ligation activities. These assays exhibit higher editing efficiencies in shorter incubation times while requiring significantly less purified editosome and 10,000-fold less ATP than the previously published full round of in vitro RNA editing assay. Moreover, modifications in the reporter ribozyme sequence enable the feasibility of multiplexing a ribozyme-based insertion/deletion editing (RIDE) assay that simultaneously surveils U-insertion and deletion editing suitable for HTS. These assays can be used to find novel chemical compounds with chemotherapeutic applications or as probes for studying the editosome machinery.

The discovery and characterization of two novel structural motifs on the carboxy-terminal domain of kinetoplastid RNA editing ligases.

Parasitic protozoans of the Trypanosoma and Leishmania species have a uniquely organized mitochondrial genome, the kinetoplast. Most kinetoplast-transcribed mRNAs are cryptic and encode multiple subunits for the electron transport chain following maturation through a uridine insertion/deletion process called RNA editing. This process is achieved through an enzyme cascade by an RNA editing catalytic complex (RECC), where the final ligation step is catalyzed by the kinetoplastid RNA editing ligases, KREL1 and KREL2. While the amino-terminal domain (NTD) of these proteins is highly conserved with other DNA ligases and mRNA capping enzymes, with five recognizable motifs, the functional role of their diverged carboxy-terminal domain (CTD) has remained elusive. In this manuscript, we assayed recombinant KREL1 in vitro to unveil critical residues from its CTD to be involved in protein-protein interaction and dsRNA ligation activity. Our data show that the α-helix (H)3 of KREL1 CTD interacts with the αH1 of its editosome protein partner KREPA2. Intriguingly, the OB-fold domain and the zinc fingers on KREPA2 do not appear to influence the RNA ligation activity of KREL1. Moreover, a specific KWKE motif on the αH4 of KREL1 CTD is found to be implicated in ligase auto-adenylylation analogous to motif VI in DNA ligases. In summary, we present in the KREL1 CTD a motif VI for auto-adenylylation and a KREPA2 binding motif for RECC integration.

Expression and functional analysis of various structural domains of tobacco topoisomerase II: To understand the mechanistic insights of plant type II topoisomerases

In contrast to bacterial, yeast and animal systems, topoisomerases (topo) from plants have not been well studied. In this report, we generated four truncated topoisomerase II (Topo II) cDNA fragments encoding different functional domains of Nicotiana tabacum topo II (NtTopoII). Each of these recombinant polypeptides was expressed alone or in combination in temperature-sensitive topoisomerase II yeast mutants. Recombinant NtTopoII with truncated polypeptides fails to target the yeast nuclei and does not rescue the temperature-sensitive phenotype. In contrast complementation was achieved with the full-length NtTopoII, which localized to the yeast nucleus. These observations suggested the presence of a potent nuclear localization signal (NLS) in the extreme C-terminal 314 amino acid residues of NtTopoII that functioned effectively in the heterologous yeast system. Biochemical characterization of purified recombinant full-length and the partial NtTopoII polypeptides revealed that the ATP-binding and hydrolysis region of NtTopoIIwas located at 413 amino acid N-terminal region and this ATPase domain is functional both when it is expressed as a separate polypeptide or as part of the holoenzyme. The present findings also revealed that all NtTopoII truncated polypeptides were detrimental for in vitro supercoiled DNA relaxation and/or DNA nicking and ligation activity. Further, we discuss the possible disruption of coordinated macromolecular interface movements and the dimer interactions in truncated NtTopoII that are required for functional topoisomerase activity.

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a β-clamp-XthA-LigA tri-component complex

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3′-hydroxyl (OH) and 5′-deoxyribose phosphate (5′-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtbβ-clamp-MtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtbβ-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbβ-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtbβ-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions.

The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of “multiple” functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.

An integrative pan-cancer analysis revealing the difference in small ring finger family of SCF E3 ubiquitin ligases.

The SCF (Skp1-cullin-F-box proteins) complex is the largest family of E3 ubiquitin ligases that mediate multiple specific substrate proteins degradation. Two ring-finger family members RBX1/ROC1 and RBX2/RNF7/SAG are small molecular proteins necessary for ubiquitin ligation activity of the multimeric SCF complex. Accumulating evidence indicated the involvement of RBX proteins in the pathogenesis and development of cancers, but no research using pan-cancer analysis for evaluating their difference has been directed previously. We investigated RBX1/2 expression patterns and the association with clinicopathological features, and survivals of cancer patients obtained from the TCGA pan-cancer data. The binding energies of RBX1/2-CUL1 complexes were preliminarily calculated by using molecular dynamics simulations. Meanwhile, we assessed their immune infiltration level across numerous databases, including TISIDB and Timer database. High expression levels of RBX1/2 were observed in most cancer types and correlated with poor prognosis of patients analyzed. Nonetheless, exceptions were observed: RBX2 expression in KICH was higher than normal renal tissues and played a detrimental role in KICH. The expression of RBX1 was not associated with the prognostic risk of KICH. Moreover, the combination of RBX1 and CUL1 expression is more stable than that of RBX2 and CUL1. RBX1/2 expression showed their own specific characteristics in tumor pathological stages and grades, copy number variation and immune components. These findings not only indicated that the difference of RBX1/2 might result in varying degrees of tumor progression, but also suggested that they might serve as biomarkers for immune infiltration in cancers, shedding new light on therapeutics of cancers.

Recombinant Expression, Purification and PEGylation of DNA Ligases

Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions. In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates. Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.

Study on activation mechanism and cleavage sites of recombinant butelase-1 zymogen derived from Clitoria ternatea

Asparagine endopeptidases (AEPs) were synthesized as a zymogen and were known to undergo pH-dependent autoproteolytic activation using their endopeptidase activity. Butelase-1, one of the few AEPs with ligation activity, can also be synthesized as a zymogen and activated at acidic pH in vitro, but the detailed activation process and potential activation sites of its zymogen are not fully understood. In this study, recombinant butelase-1 exhibited high ligation activity and ineffective endopeptidase activity, and its activities were strictly pH-dependent. The endopeptidase activity caused the activation of butelase-1 zymogen at acidic pH, which was autocatalytic, required sequential removal of C- and N-terminal pro-peptides, and was a bimolecular reaction. The pro-peptides were critical to the stability of butelase-1. Once the pro-peptides left the active domain, butelase-1 was quickly inactivated at pH 7.0. Based on the LC-MS/MS sequencing of activation products, Asp319 and Asn322 were identified as potential C-terminal pro-region hydrolysis sites of the butelase-1 zymogen, which was validated by site-directed mutagenesis. Our results provided a reasonable explanation for the self-activation of butelase-1 zymogen in vitro and provided supplementary information for the activation of AEP ligase zymogen.

Identification and Functional Characterization of Divergent 3'-Phosphate tRNA Ligase From Entamoeba histolytica.

HSPC117/RtcB, 3’-phosphate tRNA ligase, is a critical enzyme involved in tRNA splicing and maturation. HSPC117/RtcB is also involved in mRNA splicing of some protein-coding genes including XBP-1. Entamoeba histolytica, a protozoan parasite responsible for human amebiasis, possesses two RtcB proteins (EhRtcB1 and 2), but their biological functions remain unknown. Both RtcBs show kinship with mammalian/archaeal type, and all amino acid residues present in the active sites are highly conserved, as suggested by protein alignment and phylogenetic analyses. EhRtcB1 was demonstrated to be localized to the nucleus, while EhRtcB2 was in the cytosol. EhRtcB1, but not EhRtcB2, was required for optimal growth of E. histolytica trophozoites. Both EhRtcB1 (in cooperation with EhArchease) and EhRtcB2 showed RNA ligation activity in vitro. The predominant role of EhRtcB1 in tRNAIle(UAU) processing in vivo was demonstrated in EhRtcB1- and 2-gene silenced strains. Taken together, we have demonstrated the conservation of tRNA splicing and functional diversification of RtcBs in this amoebozoan lineage.

Vesicle encapsulation stabilizes intermolecular association and structure formation of functional RNA and DNA

During the origin of life, encapsulation of RNA inside vesicles is believed to have been a defining feature of the earliest cells (protocells). The confined biophysical environment provided by membrane encapsulation differs from that of bulk solution and has been shown to increase activity as well as evolutionary rate for functional RNA. However, the structural basis of the effect on RNA has not been clear. Here, we studied how encapsulation of the hairpin ribozyme inside model protocells affects ribozyme kinetics, ribozyme folding into the active conformation, and cleavage and ligation activities. We further examined the effect of encapsulation on the folding of a stem-loop RNA structure and on the formation of a triplex structure in a pH-sensitive DNA switch. The results indicate that encapsulation promotes RNA-RNA association, both intermolecular and intramolecular, and also stabilizes tertiary folding, including the docked conformation characteristic of the active hairpin ribozyme and the triplex structure. The effects of encapsulation were sufficient to rescue the activity of folding-deficient mutants of the hairpin ribozyme. Stabilization of multiple modes of nucleic acid folding and interaction thus enhanced the activity of encapsulated nucleic acids. Increased association between RNA molecules may facilitate the formation of more complex structures and cooperative interactions. These effects could promote the emergence of biological functions in an "RNA world" and may have utility in the construction of minimal synthetic cells.