Ligands as Stabilizers of G-Quadruplexes in Non-Coding RNAs.

The non-coding RNAs (ncRNA) are RNA transcripts with different sizes, structures and biological functions that do not encode functional proteins. RNA G-quadruplexes (rG4s) have been found in small and long ncRNAs. The existence of an equilibrium between rG4 and stem-loop structures in ncRNAs and its effect on biological processes remains unexplored. For example, deviation from the stem-loop leads to deregulated mature miRNA levels, demonstrating that miRNA biogenesis can be modulated by ions or small molecules. In light of this, we report several examples of rG4s in certain types of ncRNAs, and the implications of G4 stabilization using small molecules, also known as G4 ligands, in the regulation of gene expression, miRNA biogenesis, and miRNA-mRNA interactions. Until now, different G4 ligands scaffolds were synthesized for these targets. The regulatory role of the above-mentioned rG4s in ncRNAs can be used as novel therapeutic approaches for adjusting miRNA levels.

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein.

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

A ß-Hairpin Motif in the Envelope Protein E2 Mediates Receptor Binding of Bovine Viral Diarrhea Virus.

Pestivirus envelope protein E2 is crucial to virus infection and accomplishes virus-receptor interaction during entry. However, mapping of E2 residues mediating these interactions has remained unexplored. In this study, to investigate the structure-function relationship for a ß-hairpin motif exposed to the solvent in the crystal structure of bovine viral diarrhea virus (BVDV) E2, we designed two amino acidic substitutions that result in a change of electrostatic potential. First, using wild type and mutant E2 expressed as soluble recombinant proteins, we found that the mutant protein had reduced binding to susceptible cells compared to wild type and diminished ability to inhibit BVDV infection, suggesting a lower affinity for BVDV receptors. We then analyzed the effect of ß-hairpin mutations in the context of recombinant viral particles. Mutant viruses recovered from cell culture supernatant after transfection of recombinant RNA had almost completely inhibited ability to re-infect susceptible cells, indicating an impact of mutations on BVDV infectivity. Finally, sequential passaging of the mutant virus resulted in the selection of a viral population in which ß-hairpin mutations reverted to the wild type sequence to restore infectivity. Taken together, our results show that this conserved region of the E2 protein is critical for the interaction with host cell receptors.

Nucleic acids therapeutics using PolyPurine Reverse Hoogsteen hairpins.

PolyPurine Reverse Hoogsteen hairpins (PPRHs) are DNA hairpins formed by intramolecular reverse Hoogsteen bonds which can bind to polypyrimidine stretches in dsDNA by Watson:Crick bonds, thus forming a triplex and displacing the fourth strand of the DNA complex. PPRHs were first described as a gene silencing tool in vitro for DHFR, telomerase and survivin genes. Then, the effect of PPRHs directed against the survivin gene was also determined in vivo using a xenograft model of prostate cancer cells (PC3). Since then, the ability of PPRHs to inhibit gene expression has been explored in other genes involved in cancer (BCL-2, mTOR, topoisomerase, C-MYC and MDM2), in immunotherapy (SIRPα/CD47 and PD-1/PD-L1 tandem) or in replication stress (WEE1 and CHK1). Furthermore, PPRHs have the ability to target the complementary strand of a G-quadruplex motif as a regulatory element of the TYMS gene. PPRHs have also the potential to correct point mutations in the DNA as shown in two collections of CHO cell lines bearing mutations in either the dhfr or aprt loci. Finally, based on the capability of PPRHs to form triplexes, they have been incorporated as probes in biosensors for the determination of the DNA methylation status of PAX-5 in cancer and the detection of mtLSU rRNA for the diagnosis of Pneumocystis jirovecii. Of note, PPRHs have high stability and do not present immunogenicity, hepatotoxicity or nephrotoxicity in vitro. Overall, PPRHs constitute a new economical biotechnological tool with multiple biomedical applications.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Targeting replication stress response using polypurine reverse hoogsteen hairpins directed against WEE1 and CHK1 genes in human cancer cells.

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.

A novel insight in favor of structure-function relationship for 16S rRNA.

Sequences in the stem-loop part of 16S ribosomal RNA (rRNA) are considered to be crucial for predicting antibiotic resistance. Mutant sequences have been reported to be helpful in the prediction of spectinomycin resistance. It is expected that such mutations alter the 16S rRNA stem-loop conformation, which affects antibiotic binding. Metagenomic database provides 16S ribosomal DNA sequences isolated from environmental samples. Using in silico tools, we observed that the existence of specific mutation does not alter the stem-loop structure of 16S rRNA along with its three-dimensional conformation. Our observation suggests that the three-dimensional structure is a better guide to understand whether a specific mutation can cause spectinomycin resistance.

Functional pharmacogenomics and toxicity of PolyPurine Reverse Hoogsteen hairpins directed against survivin in human cells.

PolyPurine Reverse Hoogsteen (PPRH) hairpins constitute a relatively new pharmacological agent for gene silencing that has been applied for a growing number of gene targets. Previously we reported that specific PPRHs against the antiapoptotic gene survivin were able to decrease viability of PC3 prostate cancer cells by increasing apoptosis, while not acting on HUVEC non-tumoral cells. These PPRHs were efficient both in vitro and in vivo. In the present work, we performed a functional pharmacogenomics study on the effects of specific and unspecific hairpins against survivin. Incubation of PC3 cells with the specific HpsPr-C-WT led to 244 differentially expressed genes when applying the p < 0.05, FC > 2, Benjamini-Hochberg filtering. Importantly, the unspecific or control Hp-WC did not originate differentially expressed genes using the same settings. Gene Set Enrichment Analysis (GSEA) revealed that the differentially expressed genes clustered very significantly within the gene sets of Regulation of cell proliferation, Cellular response to stress, Apoptosis and Prostate cancer. Network analyses using STRING identified important interacting gene-nodes within the response of PC3 cells to treatment with the PPRH against survivin, mainly POLR2G, PAK1IP1, SMC3, SF3A1, PPARGC1A, NCOA6, UGT2B7, ALG5, VAMP7 and HIST1H2BE, the former six present in the Gene Sets detected in the GSEA. Additionally, HepG2 and 786-O cell lines were used to carry out in vitro experiments of hepatotoxicity and nephrotoxicity, respectively. The unspecific hairpin did not cause toxicity in cell survival assays (MTT) and produced minor changes in gene expression for selected genes in RT-qPCR arrays specifically developed for hepatic and renal toxicity screening.

DNA Sequence Constraints Define Functionally Active Steroid Nuclear Receptor Binding Sites in Chromatin.

Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin-interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications.

Isogenic mutations in the Moraxella catarrhalis CydDC system display pleiotropic phenotypes and reveal the role of a palindrome sequence in its transcriptional regulation.

Moraxella catarrhalis is becoming an important human respiratory tract pathogen affecting significant proportions from the population. However, still little is known about its physiology and molecular regulation. To this end, the CydDC, which is a heterodimeric ATP binding cassette transporter that has been shown to contribute to the maintenance of the redox homeostasis across the periplasm in other Gram-negative bacteria, is studied here. Amino acids multiple sequence alignments indicated that M. catarrhalis CydC is different from the CydC proteins of the bacterial species in which this system has been previously studied. These findings prompted further interest in studying this system in M. catarrhalis. Isogenic mutant in the CydDC system showed suppression in growth rate, hypersensitivity to oxidative and reductive stress and increased accumulation of intracellular cysteine levels. In addition, the growth of cydC- mutant exhibited hypersensitivity to exogenous cysteine; however, it did not display a significant difference from its wild-type counterpart in the murine pulmonary clearance model. Moreover, a palindrome was detected 94bp upstream of the cydD ORF suggesting it might act as a potential regulatory element. Real-time reverse transcription-PCR analysis showed that deletion/change in the palindrome resulted into alterations in the transcription levels of cydC. A better understanding of such system and its regulation helps in developing better ways to combat M. catarrhalis infections.

The whole chloroplast genome of wild rice (Oryza australiensis).

The whole chloroplast genome of wild rice (Oryza australiensis) is characterized in this study. The genome size is 135,224 bp, exhibiting a typical circular structure including a pair of 25,776 bp inverted repeats (IRa,b) separated by a large single-copy region (LSC) of 82,212 bp and a small single-copy region (SSC) of 12,470 bp. The overall GC content of the genome is 38.95%. 110 unique genes were annotated, including 76 protein-coding genes, 4 ribosomal RNA genes, and 30t RNA genes. Among these, 18 are duplicated in the inverted repeat regions, 13 genes contain one intron, and 2 genes (rps12 and ycf3) have two introns.

Selective Distal Enhancer Control of the Mmp13 Gene Identified through Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Genomic Deletions.

Matrix metalloproteinase 13 (Mmp13, collagenase-3) plays an essential role in bone metabolism and mineral homeostasis. It is regulated by numerous factors, including BMP-2, parathyroid hormone, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), through transcription factors such as Runt-related transcription factor 2 (RUNX2), CCAAT/enhancer-binding protein ß (C/EBPß), OSX, and vitamin D receptor (VDR). During osteoblast maturation, the basal expression of Mmp13 and its sensitivity to 1,25(OH)2D3 are strikingly increased. In this report, ChIP-sequencing analysis in mouse preosteoblasts revealed that the Mmp13 gene was probably regulated by three major enhancers located -10, -20, and -30 kb upstream of the gene promoter, occupied by activated VDR and prebound C/EBPß and RUNX2, respectively. Initially, bacterial artificial chromosome clone recombineering and traditional mutagenesis defined binding sites for VDR and RUNX2. We then employed a CRISPR/Cas9 gene editing approach to delete the -10 and -30 kb Mmp13 enhancers, a region proximal to the promoter, and VDR or RUNX2. VDR-mediated up-regulation of Mmp13 transcription was completely abrogated upon removal of the -10 kb enhancer, resulting in a 1,25(OH)2D3-directed repression of Mmp13. Deletion of either the -30 kb enhancer or RUNX2 resulted in a complete loss of basal transcript activity and a ChIP-identified destabilization of the chromatin enhancer environment and factor binding. Whereas enhancer deletions only affected Mmp13 expression, the RUNX2 deletion led to changes in gene expression, a reduction in cellular proliferation, and an inability to differentiate. We conclude that the Mmp13 gene is regulated via at least three specific distal enhancers that display independent activities yet are able to integrate response from multiple signaling pathways in a model of activation and suppression.

RNAi screening comes of age: improved techniques and complementary approaches.

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

Pocketknife tRNA hypothesis: anticodons in mammal mitochondrial tRNA side-arm loops translate proteins?

Peptide elongation proceeds by tRNA anticodons recognizing mRNA codons coding for the tRNA's cognate amino acid. Putatively, tRNAs possess three anticodons because tRNA side and anticodon-arms form similar stem-loop structures. Two lines of evidence indicate that mammal mitochondrial tRNA sidearms function as anticodons: numbers of TΨC-arm 'anticodons' matching specific cognates coevolve with that cognate's usage in mitochondrial genomes; and predicted 'tetragene' numbers, genes coded by quadruplet codons (tetracodons), coevolve with numbers of expanded anticodons in D-arms, as previously observed between tetragenes and antisense tRNA expanded anticodons. Sidearms with long stems and high GC contents contribute most to tRNA sidearm-tetragene coevolution. Results are compatible with two hypothetical mechanisms for translation by side-arms: crossovers exchange anticodon- and side-arms; tRNA sidearms are excised, aminoacylated and function as isolated stem-loop hairpins (more probable for long, respectively stable branches). Isolated sidearms would resemble recently described armless 'minimal' tRNAs. Isolated hairpins might most parsimoniously explain observed patterns. tRNA genes templating for three, rather than one functional tRNA, compress minimal genome size. Results suggest fused tRNA halves form(ed) modern tRNAs, isolated tRNA subparts occasionally translate proteins. Results confirm translational activity by antisense tRNAs, whose anticodons also coevolve with codon usages. Accounting for antisense anticodons improves results for sidearm anticodons.

Sensitive detection of DNA methyltransferase using hairpin probe-based primer generation rolling circle amplification-induced chemiluminescence.

DNA methyltransferases (MTases) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the 5-positon of cytosine in CpG islands, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Despite the development of various methods for the MTase assay, most of them are laborious and costly with poor sensitivity. Herein, we develop a highly sensitive chemiluminescence method for the MTase assay using hairpin probe-based primer generation rolling circle amplification (PG-RCA). In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate PG-RCA reaction by hybridizing with the circular DNA template, producing a large number of horseradish peroxidase-mimicking DNAzyme chains, which can catalyze the oxidation of luminal by H2O2 in the presence of hemin to yield distinct chemiluminescence signal. While in the absence of Dam MTase, neither methylation/cleavage nor PG-RCA reaction can be initiated and no chemiluminescence signal is observed. The proposed method exhibits a wide dynamic range from 0.025 to 400 U/mL and an extremely low detection limit of 1.29 × 10(-4) U/mL, which is superior to most conventional approaches for the MTase assay. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

The novel MER transposon-derived miRNAs in human genome.

MicroRNAs (miRNAs) are small RNA molecules (~20-30 nucleotides) that generally act in gene silencing and translational repression through the RNA interference pathway. They generally originate from intergenic genomic regions, but some are found in genomic regions that have been characterized such as introns, exons, and transposable elements (TE). To identify the miRNAs that are derived from palindromic MERs, we analyzed MER paralogs in human genome. The structures of the palindromic MERs were similar to the hairpin structure of miRNA in humans. Three miRNAs derived from MER96 located on chromosome 3, and MER91C paralogs located on chromosome 8 and chromosome 17 were identified in HeLa, HCT116, and HEK293 cell lines. The interactions between these MER-derived miRNAs and AGO1, AGO2, and AGO3 proteins were validated by immunoprecipitation assays. The data suggest that miRNAs derived from transposable elements could widely affect various target genes in the human genome.

Electrochemical detection of 9-hydroxyfluorene based on the direct interaction with hairpin DNA.

The direct interaction of hairpin DNA with 9-hydroxyfluorene (9-OHFLU) through hydrogen bonds was investigated by electrochemical impedance spectroscopy (EIS), UV-Vis spectroscopy and (1)H NMR spectra. Based on these results, an electrochemical hairpin DNA sensor was developed for the detection of 9-OHFLU by EIS. Upon 9-OHFLU interacting with hairpin DNA film on the gold electrodes, the charge transfer resistance (R(CT)) of the hairpin DNA film was significantly increased and remained constant after 30 min. Depending on the difference in charge transfer resistance (ΔR(CT)) before and after 9-OHFLU interaction with the hairpin DNA, 9-OHFLU could be detected with a concentration as low as 1 nM. However, only a much smaller ΔR(CT) appeared when eight selected hydroxyl polycyclic aromatic hydrocarbons (HO-PAHs) interacted with the hairpin DNA for 30 min, which demonstrated that 9-OHFLU could be discriminated from other HO-PAHs by EIS. The performance of the sensor in real lake water sample was further explored for the detection of 9-OHFLU with the detection limit of 4 nM.

CRISPR-Cas, a prokaryotic adaptive immune system, in endodontic, oral, and multidrug-resistant hospital-acquired Enterococcus faecalis.

INTRODUCTION: Microorganisms are vulnerable to invasion by mobile genetic elements such as viruses, plasmids, and transposons. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated system, or CRISPR-Cas, is an adaptive immunity system found in most archaea and many bacteria that targets and inactivates invading foreign genetic elements. Cells with CRISPR-cas are more likely to resist the invasion and uptake of foreign DNA such as viruses, plasmids, and transposons. The aims of this study were to (1) compare the occurrence of CRISPR-cas in collections of endodontic (n = 34), oral (n = 21), and multidrug-resistant hospital-acquired strains of Enterococcus faecalis (n = 23) and (2) evaluate the distribution of antibiotic resistance and virulence traits among strains without CRISPR-cas. METHODS: E. faecalis strains were screened for CRISPR1-cas and CRISPR3-cas by using polymerase chain reaction, and products were verified by DNA sequencing. Associations were investigated between the occurrence of CRISPR-cas and the expression of phenotypic traits (antibiotic resistance, gelatinase activity, bacteriocin production, hemolysin activity, and clumping response to pheromone). RESULTS: CRISPR-cas determinants were present in proportionally more endodontic (25 of 34) and oral (15 of 21) strains than hospital-acquired (9 of 23) strains (P = .01 and .04, respectively). Significant associations were found between the absence of CRISPR-cas and the presence of antibiotic resistance in strains overall (P = .04) and bacteriocin activity in endodontic strains (P = .01). CONCLUSIONS: Evidence for the presence of CRISPR-cas in the majority of endodontic and oral E. faecalis strains raises intriguing questions as to how prokaryotic immune systems might modulate interactions within the polymicrobial endodontic biofilm environment.

Persisting viral sequences shape microbial CRISPR-based immunity.

Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This 'trailer-end conservation' occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. 'Trailer-end clonality' occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining 'trailer-end conservation,' we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.

The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex.

The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.