End Overhangs Research Articles

Abstract 329: NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology

Abstract Background: Telomeres are specialized nucleoprotein structures located at the end of linear chromosomes in eukaryotic cells, and protect the ends of chromosomal DNA from degradation, end-to-end fusion and abnormal recombination. Changes in telomere length have become an important biomarker, which is closely related to cellular aging, all-cause mortality and a variety of diseases. However, there are still challenges in quantifying telomere absolute length, due to repeats of nucleotide sequence (5’-TTAGGG-3’)n in single-stranded G-rich telomeric region, and lack of a method for nucleotide separation and enrichment of individual telomeres. Methods: We developed NanoTelo, a workflow for detecting telomere absolute length based on nanopore sequencing technology. For experimental steps, a self-designed telomere adapter was first connected to the C-rich strand at the end of the chromosome by utilizing the 3' end overhang. And the 5' end of the telomere adapter was complementary to the sequencing adapter, allowing the antisense telomere chain to perform single-strand extension sequencing in the 5' to 3' direction, thus into the sub-telomeric region from the telomere end. The sequencing library was nanopore-sequenced on the QPursue-6k according to the manufacturer’s instructions. For data analysis, reads were strictly extracted (dataset B) with the telomere adapter and library structure in raw sequencing data (dataset A). The reads of dataset A and dataset B were corrected, aligned to the human reference genome (T2T-CHM13v2.0), and calculated the telomere length distribution of each chromosomal (autosomes and sex chromosomes) p/q-arm, respectively. Defined the mean telomere length of all chromosomes represented the overall telomere length, the estimated results of dataset A and dataset B were weighted to obtain the final telomere absolute length. Results: We selected 5 cell lines, which was HCT116, 293T, T24, IMR90 and WI38, and performed 2 experimental replicates for each one. We found that the estimated telomere lengths for each cell line were 3,528/3,512 bp, 4,298/4,311 bp, 2,745/2,685 bp, 4,926/4,915 bp and 4,143/4,096 bp, respectively. A comparative analysis with results from the Pacbio sequencing platform for each cell line in the Tham et al.'s paper, showed that there was no significant statistical difference (two-tailed T-test: p-value=0.98), and the mean absolute difference for estimated telomere length between the two platforms was 7.7 bp (95% CI: 5.4-10.0 bp). Conclusions: NanoTelo demonstrated the possibility of measuring telomere absolute length with a single chromosomal p/q-arm at nucleotide-level resolution. We hope that NanoTelo can not only be used as an important tool for estimating telomere length to dynamically monitor aging, but also provide strong technical support for scientific research and/or clinical application related to human anti-aging and disease intervention. Citation Format: Jidong Lang, Yanmei Chen, Zhi Yang, Beibei Huo. NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 329.

Development of Long Asymmetric siRNA Structure for Target Gene Silencing and Immune Stimulation in Mammalian Cells.

Post-transcriptional regulation of transcript abundances by RNA interference (RNAi) is a widely conserved regulatory mechanism to control cellular processes. We previously introduced an alternative siRNA structure called asymmetric siRNA (asiRNA), and showed that asiRNA exhibits comparable gene-silencing efficiency with reduced off-target effects compared with conventional siRNAs. However, to what extent the length of the guide strand affects the gene-silencing efficiency of asiRNAs is still elusive. In this study, we analyzed in detail the gene-silencing ability of asiRNAs along the guide strand length and immunostimulatory capacity of asiRNAs. We generated asiRNAs containing various guide strand lengths ranging from 25 to 29 nt, called long asiRNA (lasiRNA). We found that the gene-silencing activity of lasiRNAs decreased as the length of the guide strand increased. Nonetheless, the 3'-end overhangs that are complementary to the target gene have higher efficiency for gene silencing compared with mismatched overhangs. In addition, we found that the silencing efficiency of lasiRNAs correlates with their Ago2-binding affinity. Finally, replacing the mismatched overhang with a TLR7- or TLR9-associated immune response motif induced a toll-like receptor (TLR)-specific immune response and retained gene-silencing activity. Our findings demonstrate that lasiRNA structures can be tailored to function as bifunctional siRNA, which trigger a specific immune response combined with target gene silencing. Taken together, we anticipate that our findings provide a road map for the subsequent development of immune-stimulating lasiRNA, which bear the potential to be applied for therapeutic benefits.

A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation

We presented a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for the detection D614G mutation in S-glycoprotein of SARS-CoV-2. PEG was employed to improve the ligation efficiency of this assay by constructing a molecular crowding environment. Two hairpin probes (H1 and H2) were designed to contain 18 nt and 20 nt target binding site at their 3′ end and 5′ end, respectively. In presence of target sequence, it complemented with H1 and H2 to trigger ligation by ligase under molecular crowding condition to form ligated H1–H2 duplex. Then 3′ terminus of the H2 would be extended by DNA polymerase under isothermal conditions to form a longer extended hairpin (EHP1). 5′ terminus of EHP1 with phosphorothioate (PS) modification could form hairpin structure due to the lower Tm value. The resulting 3’ end overhang would also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains. In the circle of LSPA, long extended hairpin (EHPx) containing numerous target sequence domains was produced. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay owns an excellent linear range from 10 fM to 10 nM with a detection limit down to 4 fM. Thus, this work provides a potential isothermal amplification method for monitoring mutations in SARS-CoV-2 variants.

Trypanosoma brucei RRP44: a versatile enzyme for processing structured and non-structured RNA substrates.

Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We have determined the crystal structure of the exoribonucleolytic module of TbRRP44 in an active conformation, revealing novel details of the catalytic mechanism of the RNB domain. For the first time, the position of the second magnesium involved in the two-metal-ion mechanism was determined for a member of the RNase II family. In vitro, TbRRP44 acts preferentially on non-structured uridine-rich RNA substrates. However, we demonstrated for the first time that both TbRRP44 and its homologue from Saccharomyces cerevisiae can also degrade structured substrates without 3'-end overhang, suggesting that Rrp44/Dis3 ribonucleases may be involved in degradation of a wider panel of RNA than has been assumed. Interestingly, deletion of TbRRP44 PIN domain impairs RNA binding to different extents, depending on the type of substrate.

Gold Nanoparticle-Based Enzyme-Assisted Cyclic Amplification for the Highly-Sensitive Detection of miRNA-21

Because microRNAs (miRNAs) are biological indicators for the diagnosis, treatment, and monitoring of tumors, cancers, and other diseases, it is significant to develop a rapid, sensitive, and reliable miRNA detection platform. In this study, based on miRNA-21 detection, DNA-a with a 3′ end overhang and Texas Red fluorophore-labeled 5′ end was designed, which reacts with miRNA-21 and hybridizes with exonuclease III (Exo III), where the part connected to miRNA-21 is hydrolyzed, leaving a-DNA. At the same time, miRNA-21 is released to participate in the following reaction, to achieve cyclic amplification. a-DNA reacts with DNA-b conjugated to gold nanoparticles to achieve fluorescence quenching, with the quenching value denoted as F; additionally, after adding DNA-d and linked streptavidin immunomagnetic beads (SIBs), fluorescence recovery was achieved using DNA-c, with the recovered fluorescence recorded as F0. By comparing the difference in the fluorescence (F0 − F) between the two experiments, the amount of DNA-a hydrolyzed to produce a-DNA was established to determine the target miRNA-21 content. Under optimized conditions, by comparing the changes in the fluorescence signal, the developed strategy shows good sensitivity and repeatability, with a detection limit of 18 pM, good discriminative ability and selectivity, and promise for the early diagnosis of breast and intestinal cancers.

Dynamic modeling of a simply supported beam with an overhang mass

Abstract The aim is to derive an expression to calculate the natural frequencies and plot the mode shapes of a simply-supported beam with an overhang with an end overhang point mass by using the Euler-Bernoulli theory in the case of free transverse vibrations. The results are validated by finite element analysis. The importance of the system presented is that it can represent machine tool spindles or even machining tools like boring bars. The results are in good agreement with the results from the finite element analyses. The derived expression can be used in optimizing the value of the point mass and optimizing the support location for better performance of the system without the need to perform complex analysis to obtain the values of the natural frequencies and to plot the mode shapes.

Multifaceted regulation and functions of 53BP1 in NHEJ‑mediated DSB repair (Review).

The repair of DNA double-strand breaks (DSBs) is crucial for the preservation of genomic integrity and the maintenance of cellular homeostasis. Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism for any type of DNA DSB during the majority of the cell cycle. NHEJ defects regulate tumor sensitivity to ionizing radiation and anti-neoplastic agents, resulting in immunodeficiencies and developmental abnormalities in malignant cells. p53-binding protein 1 (53BP1) is a key mediator involved in DSB repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. 53BP1 promotes DSB repair via NHEJ and antagonizes DNA end overhang resection. At present, novel lines of evidence have revealed the molecular mechanisms underlying the recruitment of 53BP1 and DNA break-responsive effectors to DSB sites, and the promotion of NHEJ-mediated DSB repair via 53BP1, while preventing homologous recombination. In the present review article, recent advances made in the elucidation of the structural and functional characteristics of 53BP1, the mechanisms of 53BP1 recruitment and interaction with the reshaping of the chromatin architecture around DSB sites, the post-transcriptional modifications of 53BP1, and the up- and downstream pathways of 53BP1 are discussed. The present review article also focuses on the application perspectives, current challenges and future directions of 53BP1 research.

Application of the Dimeric G-Quadruplex and toehold-mediated strand displacement reaction for fluorescence biosensing of ochratoxin A

Ochratoxin A (OTA) is one of the most toxic mycotoxins that exists in various agro-products and foods. Here, a non-label and enzyme-free fluorescence biosensor for highly specific detection of OTA has been developed by the combination of toehold-mediated strand displacement reaction (TMSD) and G-quadruplex dimer/ThT (G-dimer/ThT). The DNA duplex (aptamer-IP) is composed of the anti-OTA aptamer and a single stranded initiation probe (IP). In the presence of OTA, the attachment of target to aptamer leads to the liberation of the IP, which activates the cycle TMSD amplifications of two hairpin probes (H1 and H2) accompanied by the production of numerous H1–H2 assemblies. This double-stranded H1–H2 structure results in the proximity between the 5′-end overhang tail of H1 and the 3′-end stem of H2 to liberate the pre-blocked G-dimer sequence for lighting up ThT. In addition, the method displayed a stable fluorescence emission in the high-salt media. It was successfully applied to analyze OTA in real food samples. Hence, the constructed fluorescence biosensing platform might provide a new way for OTA and other toxin analysis detection.

Label-Free Imaging of Flap Endonuclease 1 in Living Cells by Assembling Original and Multifunctional Nanoprobe.

Flap endonuclease 1 (FEN1) becomes a potential tumor marker since it is closely related to cancer occurrence and development. Here, a poly dA20-mediated nanoprobe (AuNPs-poly dA20-poly dT20) was designed for FEN1 detection. Poly dA20 segment at the 3'- end of ssDNA adsorbed on AuNPs due to its strong affinity interaction with Au (stronger than Au-S bond), while the poly dT20 segment at the 5'- end overhangs. This nanoprobe not only worked as effective fluorescence quencher but also as the original nanosubstrate of FEN1. OliGreen adsorbed on poly dT20 emits strong green fluorescence because of its high sensitivity and selectivity toward thymine. However, it is quenched on the nanoprobe. In the presence of FEN1, it recognizes the overhanging poly dT20 segment and cleaves it efficiently, turning on the fluorescence of OliGreen. This indicates that the assembled nanoprobe is an effective artificial substrate to FEN1, although it is completely different from previously reported substrates that are all composed of dsDNA with a flap strand. This proposed nanoprobe was used to detect FEN1 not only in vitro but also in vivo. The method was simple, which avoided complex labeling procedures. It had a wide linear range from 0.05 U to 2 U, with the lowest detection limit of 0.007 U. Confocal imaging can distinguish cancer cells from normal cells, demonstrating its potential in clinical diagnostic and therapeutic monitoring.

TRNA-derived G-quadruplex protects motor neurons.

Although not generally appreciated, the first 3D visualization of a nucleic acid was provided by the crystal structures of yeast phenylalanine transfer RNA (tRNA) (1, 2). [The DNA structure determined in the 1950s was a low-resolution fiber-diffraction–based model (3).] It was interesting to see that the L-shaped tRNA molecule is maintained by both Watson–Crick and non-Watson–Crick types of base interactions. These types include Hoogsteen base pairs that use the N7-C6 face of a purine to make hydrogen-bonding interactions. The scheme of the Hoogsteen base pairing also allows the formation of a planar interaction with four guanine residues, known as G-tetrad or G-quartet (Fig. 1A). Despite the fact that the G-tetrad interaction was first proposed over 50 y ago (4), demonstration of its biological significance was much later and not until it was found in the structure of telomeres (5⇓–7). The 3′-end overhang of vertebrate telomeres are rich in guanines and consist of many TTAGGG repeats that can spontaneously fold into four-stranded DNA structures with two or more G-tetrads stacking on top of each other. Functionally, formation of this unique structure—named G-quadruplex—plays a major regulatory role in telomere maintenance (8). Since then, G-quadruplex structures became widely noted to be present throughout the genome and …

Deep Sequencing Analyses of DsiRNAs Reveal the Influence of 3′ Terminal Overhangs on Dicing Polarity, Strand Selectivity, and RNA Editing of siRNAs

25/27 Base duplex RNAs that are substrates for Dicer have been demonstrated to enhance RNA interference (RNAi) potency and efficacy. Since the target sites are not always equally susceptible to suppression by small interfering RNA (siRNA), not all 27-mer duplexes that are processed into the corresponding conventional siRNAs show increased potency. Thus random designing of Dicer-substrate siRNAs (DsiRNAs) may generate siRNAs with poor RNAi due to unpredictable Dicer processing. Previous studies have demonstrated that the 3′-overhang affects dicing cleavage site and the orientation of Dicer entry. Moreover, an asymmetric 27-mer duplex having a 3′ two-nucleotide overhang and 3′-DNA residues on the blunt end has been rationally designed to obtain greater efficacy. This asymmetric structure directs dicing to predictably yield a single primary cleavage product. In the present study, we analyzed the in vitro and intracellular dicing patterns of chemically synthesized duplex RNAs with different 3′-overhangs. Consistent with previous studies, we observed that Dicer preferentially processes these RNAs at a site 21–22 nucleotide (nt) from the two-base 3′-overhangs. We also observed that the direction and ability of human Dicer to generate siRNAs can be partially or completely blocked by DNA residues at the 3′-termimi. To examine the effects of various 3′-end modifications on Dicer processing in cells, we employed Illumina Deep sequencing analyses to unravel the fates of the asymmetric 27-mer duplexes. To validate the strand selection process and knockdown capabilities we also conducted dual-luciferase psiCHECK reporter assays to monitor the RNAi potencies of both the “sense” (S) and “antisense” (AS) strands derived from these DsiRNAs. Consistent with our in vitro Dicer assays, the asymmetric duplexes were predictably processed into desired primary cleavage products of 21–22-mers in cells. We also observed the trimming of the 3′ end, especially when DNA residues were incorporated into the overhangs and this trimming ultimately influenced the Dicer-cleavage site and RNAi potency. Moreover, the observation that the most efficacious strand was the most abundant revealed that the relative frequencies of each “S” or “AS” strand are highly correlated with the silencing activity and strand selectivity. Collectively, our data demonstrate that even though the only differences between a family of DsiRNAs was the 3′ two-nuclotide overhang, dicing polarity and strand selectivity are distinct depending upon the sequence and chemical nature of this overhang. Thus, it is possible to predictably control dicing polarity and strand selectivity via simply changing the 3′-end overhangs without altering the original duplex sequence. These optimal design features of 3′-overhangs might provide a facile approach for rationally designing highly potent 25/27-mer DsiRNAs.

Oligonucleotide Bioanalysis: Sensitivity Versus Specificity

Oligonucleotide Bioanalysis: Sensitivity Versus Specificity

Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors

BackgroundSmall interfering RNAs (siRNAs) are double-stranded RNAs that effectively inhibit expression of its complimentary target mRNA. Standard siRNAs contain two nucleotide overhangs on their 3' end. While these overhangs are usually comprised of deoxythymidines (dT), it has been shown that any nucleotide can be used on the 3' end without affecting RNAi silencing.ResultsIt was recently shown that extension of the 3' end to five or eight dT molecules allows siRNAs to be effectively complexed with linear polyethylenimine (PEI), leading to enhanced cellular uptake and intracellular release. Here, we provide further evidence that only extended or 'sticky' siRNAs complexed with PEI result in significant target knockdown. However, when investigating the potential effects of these extended siRNAs on growth of human colon cancer RKO cells, we observed a dose-dependent reversal of cytotoxicity of a thymidylate synthase-targeted siRNA. In contrast, siRNAs with uridine overhangs maintained their growth inhibitory effects. We further demonstrated that dT-containing siRNAs prevented the cytotoxic effects of thymidylate synthase (TS) inhibitor compounds, such as ZD1694 and 5'-fluoro-deoxyuridine, while having no deleterious effect on cisplatin toxicity. We show that this rescue effect results from the rapid degradation of the siRNA.ConclusionsGiven that TS is an important enzyme for cell growth and proliferation and that its expression is controlled by multiple pathways, the rescue of its growth inhibitory effects may have unintended consequences. As siRNAs are being developed as therapeutic molecules, it will be important to avoid such off-target effects due to dT release. Hence, siRNAs should contain only uridine residues in their 3'-end overhangs.

Short RNA duplexes guide sequence-dependent cleavage by human Dicer.

Dicer is a member of the double-stranded (ds) RNA-specific ribonuclease III (RNase III) family that is required for RNA processing and degradation. Like most members of the RNase III family, Dicer possesses a dsRNA binding domain and cleaves long RNA duplexes in vitro. In this study, Dicer substrate selectivity was examined using bipartite substrates. These experiments revealed that an RNA helix possessing a 2-nucleotide (nt) 3'-overhang may bind and direct sequence-specific Dicer-mediated cleavage in trans at a fixed distance from the 3'-end overhang. Chemical modifications of the substrate indicate that the presence of the ribose 2'-hydroxyl group is not required for Dicer binding, but some located near the scissile bonds are needed for RNA cleavage. This suggests a flexible mechanism for substrate selectivity that recognizes the overall shape of an RNA helix. Examination of the structure of natural pre-microRNAs (pre-miRNAs) suggests that they may form bipartite substrates with complementary mRNA sequences, and thus induce seed-independent Dicer cleavage. Indeed, in vitro, natural pre-miRNA directed sequence-specific Dicer-mediated cleavage in trans by supporting the formation of a substrate mimic.

Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC.

Chemical modification of RNA duplexes can provide practical advantages for RNA interference (RNAi) triggering molecules including increased stability, safety and specificity. The impact of nucleotide modifications on Dicer processing, RISC loading and RNAi-mediated mRNA cleavage was investigated with duplexes ≥25 bp in length. It is known that dsRNAs ≥25 bp are processed by Dicer to create classic 19-bp siRNAs with 3′-end overhangs. We demonstrate that the presence of minimal modification configurations on longer RNA duplexes can block Dicer processing and result in the loading of the full-length guide strand into RISC with resultant mRNA cleavage at a defined site. These longer, modified duplexes can be highly potent gene silencers, with EC50s in the picomolar concentration range, demonstrating that Dicer processing is not required for incorporation into RISC or potent target silencing.

Multiple distinct small RNAs originate from the same microRNA precursors

BackgroundMicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes.ResultsWe report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes.ConclusionsWe demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.

Comprehensive prediction of novel microRNA targets in Arabidopsis thaliana

MicroRNAs (miRNAs) are 20–24 nt long endogenous non-coding RNAs that act as post-transcriptional regulators in metazoa and plants. Plant miRNA targets typically contain a single sequence motif with near-perfect complementarity to the miRNA. Here, we extended and applied the program RNAhybrid to identify novel miRNA targets in the complete annotated Arabidopsis thaliana transcriptome. RNAhybrid predicts the energetically most favorable miRNA:mRNA hybrids that are consistent with user-defined structural constraints. These were: (i) perfect base pairing of the duplex from nucleotide 8 to 12 counting from the 5′-end of the miRNA; (ii) loops with a maximum length of one nucleotide in either strand; (iii) bulges with no more than one nucleotide in size; and (iv) unpaired end overhangs not longer than two nucleotides. G:U base pairs are not treated as mismatches, but contribute less favorable to the overall free energy. The resulting hybrids were filtered according to their minimum free energy, resulting in an overall prediction of more than 600 novel miRNA targets. The specificity and signal-to-noise ratio of the prediction was assessed with either randomized miRNAs or randomized target sequences as negative controls. Our results are in line with recent observations that the majority of miRNA targets are not transcription factors.

RNAi‐mediated gene silencing in fishes?

The recent discovery that small regulatory RNAs have diverse roles in eukaryotic intracellular life, such as fine tuners of endogenous gene expression, as inhibitors of viral replication and as suppressors of transposon activity, is now widely recognized as a major breakthrough in the understanding of cellular biology. The pathways used by these small RNAs seem to build upon a few central mechanisms in which small double‐stranded RNAs (dsRNAs) are generated from various precursor RNAs of exogenous or endogenous origin and used for inhibiting expression of genes with sequence complementarity to the small RNA. In particular, one class of small gene‐regulatory RNAs, known as small interfering RNAs (siRNAs), has the potential for highly specific gene silencing. These are c. 21 nucleotide perfectly complementary dsRNAs with 3′ end overhangs. They are produced by cleavage of long dsRNA believed to be of viral origin. Subsequently, a large cytoplasmic protein complex known as the RNA‐induced silencing complex (RISC) uses the antisense strand of the siRNAs for targeting complementary mRNA strands followed by cleavage of these transcripts in the mechanism called RNA interference (RNAi). Accordingly, synthetic siRNAs may have a potential for treating diseases caused by intracellular pathogens like viruses by specifically targeting the expression of their molecular components. Furthermore, as target sequences need not be of viral origin, siRNAs hold a promise for the treatment of various cellular diseases and as a tool in reverse genetic studies. SiRNAs are currently exploited for such purposes in diverse organisms ranging from plants and invertebrates to human beings. But due to initial difficulties, when using RNAi for gene silencing in fishes, only a few such studies have been conducted in fishes. The purpose of this review is to inspire further experimental work.

Structural integrity of the Beta Beta Alpha-Metal finger motif is required for DNA binding and stable protein–DNA complex formation in R. KpnI

Restriction endonuclease (REase) R. KpnI from Klebsiella pneumoniae is a homodimeric enzyme, which recognizes palindromic sequence GGTAC|C and cleaves generating 4 base 3′ end overhangs. R. KpnI belongs to the HNH superfamily of nucleases, which are characterized by the presence of the ββα-Me finger motif. Structurally, this motif consists of a twisted β-hairpin followed by an α-helix, and serves as a scaffold for side chains of residues involved in co-ordination of a divalent metal ion that is required for catalysis. Homology modeling studies of R. KpnI suggested a crossover structure for the α-helix, which could possibly form dimeric interface and/or structural scaffold for the active site. We have evaluated the role of the residues present in this α-helix in intersubunit interactions and/or stabilization of the active site. We show here that mutations of residues in the α-helix lead to a loss of the enzyme activity, but not dimerization ability. Intrinsic fluorescence and circular dichroism studies revealed that the loss of function phenotype was due to the structural perturbation of the ββα-Me finger motif. The results of mutational analysis suggest that the α-helix of the ββα-Me finger of R. KpnI plays an important role for the stability of the protein–DNA complex.

Overstimulation of PrPC Signaling Pathways by Prion Peptide 106-126 Causes Oxidative Injury of Bioaminergic Neuronal Cells

Transmissible spongiform encephalopathies, also called prion diseases, are characterized by neuronal loss linked to the accumulation of PrP(Sc), a pathologic variant of the cellular prion protein (PrP(C)). Although the molecular and cellular bases of PrP(Sc)-induced neuropathogenesis are not yet fully understood, increasing evidence supports the view that PrP(Sc) accumulation interferes with PrP(C) normal function(s) in neurons. In the present work, we exploit the properties of PrP-(106-126), a synthetic peptide encompassing residues 106-126 of PrP, to investigate into the mechanisms sustaining prion-associated neuronal damage. This peptide shares many physicochemical properties with PrP(Sc) and is neurotoxic in vitro and in vivo. We examined the impact of PrP-(106-126) exposure on 1C11 neuroepithelial cells, their neuronal progenies, and GT1-7 hypothalamic cells. This peptide triggers reactive oxygen species overflow, mitogen-activated protein kinase (ERK1/2), and SAPK (p38 and JNK1/2) sustained activation, and apoptotic signals in 1C11-derived serotonergic and noradrenergic neuronal cells, while having no effect on 1C11 precursor and GT1-7 cells. The neurotoxic action of PrP-(106-126) relies on cell surface expression of PrP(C), recruitment of a PrP(C)-Caveolin-Fyn signaling platform, and overstimulation of NADPH-oxidase activity. Altogether, these findings provide actual evidence that PrP-(106-126)-induced neuronal injury is caused by an amplification of PrP(C)-associated signaling responses, which notably promotes oxidative stress conditions. Distorsion of PrP(C) signaling in neuronal cells could hence represent a causal event in transmissible spongiform encephalopathy pathogenesis.