Complementary Target RNAs Research Articles

Highly sensitive detection of rare mutant alleles by combining argonaute-based enrichment and XNA-PCR.

e14605 Background: Characterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early disease detection, genotyping, and personalized therapy. Detection of alleles of clinical interest is often challenged by their low concentration and sequence homology with the much more abundant wildtype nucleic acids. Methods: Argonuate (Ago) from the thermophilic bacterium Thermus thermophilus ( TtAgo) utilizes short DNA guides to specifically cleave complementary DNA and RNA targets. We found that under optimized conditions, TtAgo cleaves DNA and RNA complementary to the guide DNA with high efficiency, but spares nucleic acids with a single nucleotide mismatch at and around its catalytic site with high sensitivity. Based on these findings, we designed a new multiplexed enrichment assay, dubbed NAVIGATER (Nucleic Acid enrichment Via DNA Guided Argonaute from Thermus thermophilus), that utilizes TtAgo, to specifically cleave perfectly complementary DNA and RNA while sparing alleles of interest. Results: NAVIGATER greatly increases the fractions of rare mutant alleles with single nucleotide precision enhancing the sensitivity of downstream detection methods such as XNA-PCR. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and over ten-fold improved sensitivity of XNA-PCR, enabling multiplex detection of KRAS and EGFR mutants at 0.01% fractions. Conclusions: NAVIGATER has important advantages over other mutant allele enrichment assays such as the ones based on CRISPR-Cas. It does not require the target to contain a protospacer-adjacent motif; is a true (turnover) catalyst; can cleave both DNA and associated exosomal RNA targets, improving sensitivity; and can operate at elevated temperatures for higher selectivity and compatibility with detection schemes.

Small RNAs from Seed to Mature Plant

Small regulatory RNAs (sRNAs) are being used by plants to direct gene expression. In plants, different classes of sRNAs exist based on their biogenesis and origin. Among them, small interfering RNAs (siRNAs) and microRNAs (miRNAs) chiefly mask post-transcriptional gene expression. Initiation of translational inhibition or degradation requires the binding of miRNAs with the complementary messenger RNA (mRNA) target sites. From seed germination to plant formation, current studies report that sRNAs have a significant role in various plant developmental phases. Moreover, these sRNAs have been engaged throughout the plant evolutionary pathway, playing an active role in the shaping and functioning of various plant organs. Given the importance of sRNAs in the plant development and life cycle, this review article elaborates on the critical involvement of sRNAs in different phases of plant life starting from the seed and progressing towards the mature plant.

Dynamics of Cas10 Govern Discrimination between Self and Non-self in Type III CRISPR-Cas Immunity

Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a typeIII-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity invivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small noncoding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.

α-l-Threose Nucleic Acids as Biocompatible Antisense Oligonucleotides for Suppressing Gene Expression in Living Cells.

Because of the chemical simplicity of α-l-threose nucleic acid (TNA) and its ability to exchange genetic information between itself and RNA, it has attracted significant interest as the RNA ancestor. We herein explore the biological properties and evaluate the potency of sequence-designed TNA polymers to suppress the gene expression in living environments. We found that sequence-specific TNA macromolecules exhibit strong affinity and specificity toward the complementary RNA targets, are highly biocompatible and nontoxic in a living cell system, and readily enter a number of cell lines without using transfecting agents. Particularly, TNA exhibited much stronger enzymatic resistance toward fetal bovine serum or human serum as compared to traditional antisense oligonucleotides, which means that the intrinsic structure of TNA is thoroughly resistant to biological degradation. Importantly, the efficacy of the TNA molecule with green fluorescent protein (GFP) target sequence (anti-GFP TNAs) as antisense agents was first demonstrated in living cells in which these polymers revealed high antisense activity in terms of the degree of inhibition of GFP gene expression. The GFP gene inhibition studies in HeLa and HEK293 cells characterize sequence-controlled TNA as a functional biomaterial and a valuable alternative to traditional antisense oligonucleotides such as peptide nucleic acids, phosphorodiamidate morpholino oligomers, and locked nucleic acids for a wide range of applications in drug discovery and life science research. Additionally, we also first reported the cost-efficient approach to synthesize the four TNA phosphoramidite monomers using 2-cyanoethyl N, N, N', N'-tetraisopropylphosphoramidite as a key reagent. Furthermore, by increasing the frequency of the deblocking and coupling reactions together with extending their reaction time in each synthesis cycle, sequence-controlled TNAs can be easily synthesized in a quantitative yield and high purity.

Electrochemical detection of microRNAs by graphene oxide modified disposable graphite electrodes

Graphene oxide (GO) modified disposable pencil graphite electrodes (PGEs) were developed for the first time in the present study for miRNA detection by measuring the guanine oxidation signal. GO modified PGEs were used as solid phase to immobilize amino linked DNA probe and then the complementary target RNA sequence (miRNA-34a) was recognized. The electrochemical behaviour of the GO modified electrodes was investigated by using electrochemical impedance spectroscopy (EIS) and microscopic characterization was performed by scanning electron microscopy (SEM). The optimization studies and detection of hybridization between miRNA-34a target and its complementary DNA probe was performed by differential pulse voltammetry (DPV). The selectivity of our assay was tested in the presence of non-complementary miRNA sequence. A good discrimination could be achieved compared to the results obtained with the full match hybridization of probe with its RNA target miRNA-34a target against other miRNA sequences; miRNA-16 and miRNA-155.

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Human Argonaute3 has slicer activity.

Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3 and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes that bind target mRNAs and silence gene expression through translational repression and deadenylation but not cleavage. Here, we report that recombinant AGO3 loaded with miR-20a cleaves complementary target RNAs, whereas AGO3 loaded with let-7a, miR-19b or miR-16 does not, indicating that AGO3 has slicer activity but that this activity depends on the guide RNA. Our cleavage assays using chimeric guides revealed the significance of seed sequence for AGO3 activity, which depends specifically on the sequence of the post-seed. Unlike AGO2, target cleavage by AGO3 requires both 5′- and 3′-flanking regions. Our 3.28 Å crystal structure shows that AGO3 forms a complete active site mirroring that of AGO2, but not a well-defined nucleic acid-binding channel. These results demonstrating that AGO3 also has slicer activity but with more intricate substrate requirements, explain the observation that AGO3 has retained the necessary catalytic residues throughout its evolution. In addition, our structure inspires the idea that the substrate-binding channel of AGO3 and consequently its cellular function, may be modulated by accessory proteins.

Towards Improved Oligonucleotide Therapeutics Through Faster Target Binding Kinetics.

When used as inhibitors of gene expression in vivo, oligonucleotides require modification of their structures to boost their binding affinity for complementary target RNAs. To date, hundreds of modifications have been designed and tested but few have proven to be useful. Among those investigated are mono- and polyamino-groups. These are positively charged at physiological pH and have been appended to oligonucleotides in an effort to reduce electrostatic repulsion during hybridization to RNAs, but have generally shown relatively minor benefits to binding. We conjugated spermine to uracils in oligonucleotides via a triazole linker so that the polyamine fits in the major groove of a subsequently formed RNA-duplex. The modifications produced large increases in target-binding affinity of the oligonucleotides. Using surface plasmon resonance-based assays, we showed that the increases derived mainly from faster annealing (kon ). We propose that the spermine fragments play a similar role to that of natural polyamines during oligonucleotide-target interactions in cells, and may be advantageous for oligonucleotides that operate catalytic mechanisms.

Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3′-exonuclease stability. According to CD-spectroscopy studies G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.

Non-canonical targets destabilize microRNAs in human Argonautes.

Although much is known about microRNA (miRNA) biogenesis and the mechanism by which miRNAs regulate their targets, little is known about the regulation of miRNA stability. Mature miRNAs are stabilized by binding to Argonaute (Ago) proteins, the core components of the RNA-induced silencing complex (RISC). Recent studies suggest that interactions between miRNAs and their highly complementary target RNAs promote release of miRNAs from Ago proteins, and this in turn can lead to destabilization of miRNAs. However, the physiological triggers of miRNA destabilization with molecular mechanisms remain largely unknown. Here, using an in vitro system that consists of a minimal human Ago2–RISC in HEK293T cell lysates, we sought to understand how miRNAs are destabilized by their targets. Strikingly, we showed that miRNA destabilization is dramatically enhanced by an interaction with seedless, non-canonical targets. We then showed that this process entails not only unloading of miRNAs from Ago, but also 3΄ end destabilization of miRNAs occurred within Ago. Furthermore, our mutation analysis indicates that conformational changes in the hinge region of the Ago PAZ domain are likely to be the main driving force of the miRNA destabilization. Our collective results suggest that non-canonical targets may provide a stability control mechanism in the regulation of miRNAs in humans.

Covering the Cover

Covering the Cover

Kinetic Analysis of Target RNA Binding and Slicing by Human Argonaute 2 Protein.

Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels. Here, we present techniques to kinetically characterize recombinant Argonaute 2-mediated guide and target binding as well as target RNA slicing. We focus on fluorescence-based steady-state and in particular pre-steady-state techniques to unravel mechanistic details. Furthermore, we describe a cleavage assay to analyze Argonaute 2-mediated slicing using radioactively labeled target strands.

Northern Blotting Techniques for Small RNAs.

Cells have evolved intricate RNA-directed mechanisms that destroy viruses, silence transposons, and regulate gene expression. These nucleic acid surveillance and gene silencing mechanisms rely upon the selective base-pairing of ~19-25 nt small RNAs to complementary RNA targets. This chapter describes northern blot hybridization techniques for the detection of such small RNAs. Blots spiked with synthetic standards are used to illustrate the detection specificity and sensitivity of DNA oligonucleotide probes. Known endogenous small RNAs are then analyzed in samples prepared from several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Oryza sativa, Zea mays, and Physcomitrella patens, as well as from the animals Drosophila melanogaster and Mus musculus. Finally, the value of northern blotting for dissecting small RNA biogenesis is shown using an example of virus infection in A. thaliana.

Identification of factors involved in target RNA-directed microRNA degradation.

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.

Two wavelength-shifting molecular beacons for simultaneous and selective imaging of vesicular miRNA-21 and miRNA-31 in living cancer cells.

Two molecular beacons were designed as complementary fluorescent imaging probes for miRNA-21 and miRNA-31. Both beacons were prepared by a combination of solid-phase protocol and Cu(i)-catalyzed cycloaddition chemistry. The four photostable and bright fluorophores were attached to 2'-positions in the stem part of the two beacons. One beacon was labeled by a green-to-red emitting and the other by a blue-to-yellow emitting energy transfer pair. This two by two combination yields the four color emission readout. In vitro experiments demonstrate rapid and highly selective opening of both molecular beacons upon addition of the complementary target RNA and excellent green : red and blue : yellow emission color contrasts. Confocal microscopy of selected cancer cell lines provides evidence that a four color imaging of versicular miRNA-21 and miRNA-31 can be achieved both selectively and simultaneously upon transfection by the beacons, and that the fluorescent readouts track well with miRNA levels determined by PCR.

DNA targeting by the type I-G and type I-A CRISPR-Cas systems of Pyrococcus furiosus.

CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated.

Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition.

Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids.

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations.

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus.

CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5′ ends (8-nt repeat-derived 5′ tag sequences) but heterogeneous 3′ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3′ end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.