Solid-phase translation and RNA-protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA

A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA-protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA-protein fusion is ligated to the 3' ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA-protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of 'RNA chip-to-protein chip' using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution.     

Visualization and characterization of RNA–protein interactions in living cells

RNA–protein interactions are the structural and functional basis of significant numbers of RNA molecules. RNA–protein interaction assays though, still mainly depend on biochemical tests in vitro. Here, we establish a convenient and reliable RNA fluorescent three-hybrid (rF3H) method to detect/interrogate the interactions between RNAs and proteins in cells. A GFP tagged highly specific RNA trap is constructed to anchor the RNA of interest to an artificial or natural subcellular structure, and RNA–protein interactions can be detected and visualized by the enrichment of RNA binding proteins (RBPs) at these structures. Different RNA trapping systems are developed and detection of RNA–protein complexes at multiple subcellular structures are assayed. With this new toolset, interactions between proteins and mRNA or noncoding RNAs are characterized, including the interaction between a long noncoding RNA and an epigenetic modulator. Our approach provides a flexible and reliable method for the characterization of RNA–protein interactions in living cells.     

RNA pull-down confocal nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-synuclein levels

RNA–protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA–protein interactions have shown promising effects on some diseases that are previously regarded as ‘incurable’. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA–protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7–1. Importantly, miR-7′s primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson’s disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson’s disease as well as provides a novel methodology to search for modulators of RNA–protein interaction.     

Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip

Taking advantage of the "open" nature of cell-free protein synthesis, this study investigated the direct analysis of protein expression using a surface plasmon resonance sensor. During the on-chip incubation of the reaction mixture for cell-free protein synthesis, the expressed protein molecules were immobilized onto the surface of the chip, giving rise to a sensorgram signal, which enabled on-line monitoring of protein expression. In addition, we found that the expression of the aggregation-prone proteins could be effectively monitored. The ability to monitor these proteins was most likely through the instant isolation of the expressed protein molecules onto the solid surface of the chip.     

Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA–protein complexes

A set of >300 nonredundant high-resolution RNA–protein complexes were rigorously searched for π-contacts between an amino acid side chain (W, H, F, Y, R, E and D) and an RNA nucleobase (denoted π–π interaction) or ribose moiety (denoted sugar–π). The resulting dataset of >1500 RNA–protein π-contacts were visually inspected and classified based on the interaction type, and amino acids and RNA components involved. More than 80% of structures searched contained at least one RNA–protein π-interaction, with π–π contacts making up 59% of the identified interactions. RNA–protein π–π and sugar–π contacts exhibit a range in the RNA and protein components involved, relative monomer orientations and quantum mechanically predicted binding energies. Interestingly, π–π and sugar–π interactions occur more frequently with RNA (4.8 contacts/structure) than DNA (2.6). Moreover, the maximum stability is greater for RNA–protein contacts than DNA–protein interactions. In addition to highlighting distinct differences between RNA and DNA–protein binding, this work has generated the largest dataset of RNA–protein π-interactions to date, thereby underscoring that RNA–protein π-contacts are ubiquitous in nature, and key to the stability and function of RNA–protein complexes.     

Putative intermediary stages for the molecular evolution from a ribozyme to a catalytic RNP

A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA–protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA–RNA interaction in the starting ribozyme is replaced with an RNA–protein interaction via two intermediary stages. At each stage, the original RNA–RNA interaction and a newly introduced RNA–protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA–RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity.     

Genome-Wide Double-Stranded RNA Sequencing Reveals the Functional Significance of Base-Paired RNAs in Arabidopsis

The functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation require formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we marry classical nuclease-based structure mapping techniques with high-throughput sequencing technology to interrogate all base-paired RNA in Arabidopsis thaliana and identify ∼200 new small (sm)RNA–producing substrates of RNA–DEPENDENT RNA POLYMERASE6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. Although our methodology reveals the pairing status of RNA molecules in the absence of cellular proteins, previous studies have demonstrated that structural information obtained for RNAs in solution accurately reflects their structure in ribonucleoprotein complexes. Furthermore, our identification of RNA–DEPENDENT RNA POLYMERASE6 substrates and conserved functional RNA domains within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs using this approach strongly suggests that RNA molecules are correctly folded into their secondary structure in solution. Overall, our findings highlight the importance of base-paired RNAs in eukaryotes and present an approach that should be widely applicable for the analysis of this key structural feature of RNA.     

The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function

Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES–40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES–RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA–protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES–RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S–3′ UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES–RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.     

IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.     

Directed evolution of orthogonal RNA–RBP pairs through library-vs-library in vitro selection

RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA–RBP pairs from natural components remains challenging while such synthetic RNA–RBP pairs could significantly expand the RNA–RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 10¹² unique RNA sequences and 4.0 × 10⁸ unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA–RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA–RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.     

Single Domain Antibodies as a Powerful Tool for High Quality Surface Plasmon Resonance Studies

Single domain antibodies are recombinantly expressed functional antibodies devoid of light chains. These binding elements are derived from heavy chain antibodies found in camelids and offer several distinctive properties for applications in biotechnology such as small size, stability, solubility, and expression in high yields. In this study we demonstrated the potential of using single domain antibodies as capturing molecules in biosensing applications. Single domain antibodies raised against green fluorescent protein were anchored onto biosensor surfaces by using several immobilization strategies based on Ni²⁺:nitrilotriacetic acid-polyhistidine tag, antibody-antigen, biotin-streptavidin interactions and amine-coupling chemistry. The interaction with the specific target of the single domain antibodies was characterized by surface plasmon resonance. The immobilized single domain antibodies show high affinities for their antigens with K_D = 3–6 nM and outperform other antibody partners as capturing molecules facilitating also the data analysis. Furthermore they offer high resistance and stability to a wide range of denaturing agents. These unique biophysical properties and the production of novel single domain antibodies against affinity tags make them particularly attractive for use in biosensing and diagnostic assays.     

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond

Single-stranded regions in RNA secondary structure are important for RNA–RNA and RNA–protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals ‘well-determined’ single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit β-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA–RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.     

Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.     

Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment

Reoviruses are important human, animal and plant pathogens having 10–12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA–protein and RNA–RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA–RNA interactions between genomic precursors during segment assortment and packaging.     

RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct

RNA–RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA–RNA interactions can involve complementary Watson–Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA–RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem–loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem–loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem–loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem–loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop–loop ‘kissing’ motif. High affinity binding to the other structural motifs in the decoding region were not observed.