Time-resolved structural analysis of an RNA-cleaving DNA catalyst.

The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.

Viroid research and its significance for RNA technology and basic biochemistry.

Viroids were described 47 years ago as the smallest RNA molecules capable of infecting plants and autonomously self-replicating without an encoded protein. Work on viroids initiated the development of a number of innovative methods. Novel chromatographic and gelelectrophoretic methods were developed for the purification and characterization of viroids; these methods were later used in molecular biology, gene technology and in prion research. Theoretical and experimental studies of RNA folding demonstrated the general biological importance of metastable structures, and nuclear magnetic resonance spectroscopy of viroid RNA showed the partially covalent nature of hydrogen bonds in biological macromolecules. RNA biochemistry and molecular biology profited from viroid research, such as in the detection of RNA as template of DNA-dependent polymerases and in mechanisms of gene silencing. Viroids, the first circular RNA detected in nature, are important for studies on the much wider spectrum of circular RNAs and other non-coding RNAs.

Molecular Features and Metal Ions That Influence 10-23 DNAzyme Activity.

Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.

Inability of DNAzymes to cleave RNA in vivo is due to limited Mg[Formula: see text] concentration in cells.

Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10-23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg[Formula: see text] bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg[Formula: see text] ions more strongly and already stoichiometric amounts of Mg[Formula: see text] and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg[Formula: see text] concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg[Formula: see text] concentrations than that available inside the cell.

Viroids, Satellite RNAs and Prions: Folding of Nucleic Acids and Misfolding of Proteins.

Theodor ("Ted") Otto Diener (* 28 February 1921 in Zürich, Switzerland; † 28 March 2023 in Beltsville, MD, USA) pioneered research on viroids while working at the Plant Virology Laboratory, Agricultural Research Service, USDA, in Beltsville. He coined the name viroid and defined viroids' important features like the infectivity of naked single-stranded RNA without protein-coding capacity. During scientific meetings in the 1970s and 1980s, viroids were often discussed at conferences together with other "subviral pathogens". This term includes what are now called satellite RNAs and prions. Satellite RNAs depend on a helper virus and have linear or, in the case of virusoids, circular RNA genomes. Prions, proteinaceous infectious particles, are the agents of scrapie, kuru and some other diseases. Many satellite RNAs, like viroids, are non-coding and exert their function by thermodynamically or kinetically controlled folding, while prions are solely host-encoded proteins that cause disease by misfolding, aggregation and transmission of their conformations into infectious prion isoforms. In this memorial, we will recall the work of Ted Diener on subviral pathogens.

Development and Implementation of an Internal Quality Control Sample to Standardize Oligomer-Based Diagnostics of Alzheimer's Disease.

Protein misfolding and aggregation are pathological hallmarks of various neurodegenerative diseases. In Alzheimer's disease (AD), soluble and toxic amyloid-ß (Aß) oligomers are biomarker candidates for diagnostics and drug development. However, accurate quantification of Aß oligomers in bodily fluids is challenging because extreme sensitivity and specificity are required. We previously introduced surface-based fluorescence intensity distribution analysis (sFIDA) with single-particle sensitivity. In this report, a preparation protocol for a synthetic Aß oligomer sample was developed. This sample was used for internal quality control (IQC) to improve standardization, quality assurance, and routine application of oligomer-based diagnostic methods. We established an aggregation protocol for Aß1-42, characterized the oligomers by atomic force microscopy (AFM), and assessed their application in sFIDA. Globular-shaped oligomers with a median size of 2.67 nm were detected by AFM, and sFIDA analysis of the Aß1-42 oligomers yielded a femtomolar detection limit with high assay selectivity and dilution linearity over 5 log units. Lastly, we implemented a Shewhart chart for monitoring IQC performance over time, which is another important step toward quality assurance of oligomer-based diagnostic methods.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

In vitro conversion and seeded fibrillization of posttranslationally modified prion protein.

The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrP(C) into an aggregated, ß-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.

Structural changes of membrane-anchored native PrP(C).

Misfolding and subsequent aggregation of endogenous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular beta-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases.

Mechanisms of prion protein assembly into amyloid.

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP(C)) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP(Sc)) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.

Characterization of plant miRNAs and small RNAs derived from potato spindle tuber viroid (PSTVd) in infected tomato.

To defend against invading pathogens, plants possess RNA silencing mechanisms involving small RNAs (miRNAs, siRNAs). Also viroids - plant infectious, non-coding, unencapsidated RNA - cause the production of viroid-specific small RNAs (vsRNA), but viroids do escape the cytoplasmic silencing mechanism. Viroids with minor sequence variations can produce different symptoms in infected plants, suggesting an involvement of vsRNAs in symptom production. We analyzed by deep sequencing the spectrum of vsRNAs induced by the PSTVd strain AS1, which causes strong symptoms such as dwarfing and necrosis upon infection of tomato plants cv Rutgers. Indeed, vsRNAs found with highest frequency mapped to the pathogenicity-modulating domain of PSTVd, supporting an involvement of vsRNAs in symptom production. Furthermore, in PSTVd AS1-infected plants the accumulation of some endogenous miRNAs, which are involved in leaf development via regulation of transcription factors, is suppressed. The latter finding supports the hypothesis that a miRNA-dependent (mis)regulation of transcription factors causes the viroid symptoms.

Prion detection by an amyloid seeding assay.

Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP(Sc), the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was approximately 1 fg. Some prion strains consist largely of protease-sensitive PrP(Sc) (sPrP(Sc)), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP(Sc) and protease-resistant PrP(Sc) that does not rely on protease digestion or immunodetection.

Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity.

The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrP(Sc), associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrP(Sc) that recognizes both PrP(C) and PrP(Sc). This scFv fragment had a dissociation constant (K(D)) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrP(Sc) but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrP(Sc) and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs.

Spontaneous and BSE-prion-seeded amyloid formation of full length recombinant bovine prion protein.

The conversion of the cellular isoform of the prion protein into the pathogenic isoform PrP(Sc) is the key event in prion diseases. The disease can occur spontaneously genetically or by infection. In earlier studies we presented an in vitro conversion system which simulates the structural transition in recPrP by varying low concentrations of SDS at constant NaCl. In the present study we adopted the conversion system from experimental Scrapie in hamster to bovine recPrP and generated amyloid fibrils. The intermediate state which is optimal for fibril formation is a soluble, beta-rich state. The system was extended using BSE-prions as seeds and led to an acceleration of fibril formation by orders of magnitude. This seeded amyloid formation assay avoids any PK-treatment, is therefore able to detect even PK-sensitive PrP(Sc) and does not require cellular components.

A highly sensitive diagnostic assay for aggregate-related diseases, including prion diseases and Alzheimer's disease.

Prion diseases, Alzheimer's disease, and Parkinson's disease are age-related neurodegenerative diseases that are characterized by the formation of protein aggregates during the progress of the disease. Although it is still not known whether these aggregates are causative for, or symptoms of, the disease. Many studies show that aggregates or even oligomers of the according proteins are neurotoxic and thus may lead to neurodegeneration. To understand disease-associated or causative mechanisms in respect to protein aggregation, an ultrasensitive tool to quantify these disease-related aggregates is required. In this study we introduce a specificity-enhanced version of surface-FIDA as an approach to count even single aggregates in tissue homogenate and body liquids.

Aggregation and amyloid fibril formation of the prion protein is accelerated in the presence of glycogen.

Prion diseases like Creutzfeldt-Jakob disease in humans or scrapie in sheep and goats are infectious neurodegenerative diseases. Their infectious agent, called prion, is composed mainly of aggregated and misfolded prion protein and non-proteinaceous components. An example of such a common non-proteinaceous secondary component of natural prions is the polysaccharide scaffold. We studied the influence of such a polysaccharide on the conformational transition of PrP applying an in vitro conversion system. Here we report that glycogen supports and accelerates PrP amorphous aggregation similar to seeded aggregation and leads to co-aggregates. Furthermore, PrP fibril formation was highly accelerated in the presence of glycogen.

An ultrasensitive assay for diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the most common cause of dementia. Aging is among the most significant risk factors. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble beta-amyloid peptide (Abeta) aggregates in the patient's brain tissue. We have developed an ultrasensitive assay for early and non-invasive diagnosis of AD. This highly specific and sensitive assay uses fluorescence correlation spectroscopy (FCS) and is sensitive enough to detect even single aggregates in body fluids of AD patients. We investigate the correlation of aggregated Abeta concentrations in body fluids with clinical symptoms of AD.

Single particle detection of Abeta aggregates associated with Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Today, AD can be diagnosed with certainty only post-mortem, by histopathologic staining of Abeta plaques and neurofibrillary tangles in brain tissue sections. We have developed an ultra-sensitive assay potentially suitable for early and non-invasive diagnosis of AD. This highly specific and sensitive assay uses fluorescence correlation spectroscopy (FCS) and is sensitive enough to detect even single aggregates in body fluids of AD patients. First results show a clear distinction between AD diseased people and non-demented controls by analysing cerebrospinal fluids (CSF) by confocal scanning of surface captured Abeta aggregates and subsequent two-dimensional fluorescence intensity distribution analysis.

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA).

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Detection and analysis of hairpin II, an essential metastable structural element in viroid replication intermediates.

In (-)-stranded replication intermediates of the potato spindle tuber viroid (PSTVd) a thermodynamically metastable structure containing a specific hairpin structure (HP II) has been proposed to be essential for viroid replication. In the present work a method was devised allowing the direct detection of the HP II structure in vitro and in vivo using a biophysical approach. An RNA oligonucleotide was constructed which specifically binds to the HP II loop region in transient (-)-strand intermediates. Analysis of the resulting oligonucleotide/HP II complexes on temperature-gradient gels enabled us to follow the formation of HP II during in vitro transcription by T7 RNA polymerase. Moreover, we were able to demonstrate the formation of HP II during viroid replication in potato (Solanum tuberosum) cells.