Insights into innate immune activation via PS-ASO-protein-TLR9 interactions.

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary-in part-as a function of 2'modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO-protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.

NAT10 and DDX21 Proteins Interact with RNase H1 and Affect the Performance of Phosphorothioate Oligonucleotides.

RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging.

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.

Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translation.

Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.

Oligonucleotide Phosphorothioates Enter Cells by Thiol-Mediated Uptake.

Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non-modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA-approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol-mediated uptake. The transient adaptive network produced by dynamic covalent pseudo-disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.

Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

The lymphatic system is a network of ducts that transports fluid, proteins, and immune cells from different organs around the body. Lymph nodes provide pit stops at hundreds of points along this network where immune cells reside, and lymph fluid can be filtered and cleaned. When pathogens, such as viruses or bacteria, enter the body during an infection, fragments of their proteins can get swept into the lymph nodes. These pathogenic proteins or protein fragments activate resident immune cells and kickstart the immune response. Vaccines are designed to mimic this process by introducing isolated pathogenic proteins in a controlled way to stimulate similar immune reactions in lymph nodes. Once an infection has been cleared by the immune system, or a vaccination has triggered the immune system, most pathogenic proteins get cleared away. However, a small number of pathogenic proteins remain in the lymph nodes to enable immune cells to respond more strongly and quickly the next time they see the same pathogen. Yet it is largely unclear how much protein remains for training and how or where it is all stored. Current techniques are not sensitive or long-lived enough to accurately detect and track these small protein deposits over time. Walsh, Sheridan, Lucas, et al. have addressed this problem by developing biological tags that can be attached to the pathogenic proteins so they can be traced. These tags were designed so the body cannot easily break them down, helping them last as long as the proteins they are attached to. Walsh, Sheridan, Lucas et al. tested whether vaccinating mice with the tagged proteins allowed the proteins to be tracked. The method they used was designed to identify individual cell types based on their genetic information along with the tag. This allowed them to accurately map the complex network of cells involved in storing and retrieving archived protein fragments, as well as those involved in training new immune cells to recognize them. These results provide important insights into the protein archiving system that is involved in enhancing immune memory. This may help guide the development of new vaccination strategies that can manipulate how proteins are archived to establish more durable immune protection. The biological tags developed could also be used to track therapeutic proteins, allowing scientists to determine how long cancer drugs, antibody therapies or COVID19 anti-viral agents remain in the body. This information could then be used by doctors to plan specific and personalized treatment timetables for patients.

Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins.

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2'-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein-protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect.

mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5' cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the ß-phosphate (ß-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo. However, the molecular basis for the beneficial "thio-effect" remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the ß-O-to-S or -Se substitution on the interaction with eIF4E. We determined the SP/RP configurations of ß-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the ß-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both ß-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the "thio-effect" was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions.

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The Tm values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5'→3' (T5 Exo) and 3'→5' direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents.

Recent progress in understanding phosphorothioate antisense oligonucleotide (PS-ASO) interactions with proteins has revealed that proteins play deterministic roles in the absorption, distribution, cellular uptake, subcellular distribution, molecular mechanisms of action, and toxicity of PS-ASOs. Similarly, such interactions can alter the fates of many intracellular proteins. These and other advances have opened new avenues for the medicinal chemistry of PS-ASOs and research on all elements of the molecular pharmacology of these molecules. These advances have recently been reviewed. In this Perspective article, we summarize some of those learnings, the general principles that have emerged, and a few of the exciting new questions that can now be addressed.

Defense Mechanism of Phosphorothioated DNA under Peroxynitrite-Mediated Oxidative Stress.

DNA phosphorothioation (PT) exists in many pathogenic bacteria; however, the mechanism of PT-DNA resistance to the immune response is unclear. In this work, we meticulously investigated the peroxynitrite (PN) tolerance using PT-bioengineered E. coli strains. The in vivo experiment confirms that the S+ strain survives better than the S- strain under moderately oxidative stress. The LCMS, IC, and GCMS experiments demonstrated that phosphorothioate partially converted to phosphate, and the byproduct included sulfate and elemental sulfur. When O,O-diethyl thiophosphate ester (DETP) was used, the reaction rate k1 was determined to be 4.3 ± 0.5 M-1 s-1 in the first-order for both phosphorothioate and peroxynitrite at 35 °C and pH of 8.0. The IC50 values of phosphorothioate dinucleotides are dramatically increased by 400-700-fold compared to DETP. The SH/OH Yin-Yang mechanism rationalizes the in situ DNA self-defense against PN-mediated oxidative stress at the extra bioenergetic cost of DNA modification.

Phosphorothioate modified oligonucleotide-protein interactions.

Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.

In vitro metabolism of 2'-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry.

Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC-tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3'-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5'-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.

An in vitro DNA phosphorothioate modification reaction.

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from Escherichia coli. We determined that the in vitro process of PT was oxygen sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM) and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. Adenosine triphosphate (ATP) promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1.

The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.

Studying the Biotransformation of Phosphorothioate-Containing Oligonucleotide Drugs by LC-MS.

Across the pharmaceutical industry, there is increasing interest and need to investigate the biotransformation of oligonucleotide drugs. The method of choice is high-resolution mass spectrometry due to its unmet sensitivity and specificity.Here, we describe a method developed and applied in our laboratory studying the biotransformation of phosphorothioate-containing oligonucleotide drugs. This method is based on capillary flow liquid chromatography with column switching coupled to high-resolution mass spectrometry.

Investigating the interactions between DNA and DndE during DNA phosphorothioation.

The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild-type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE-DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding.

Determination of oligonucleotide deamination by high resolution mass spectrometry.

A novel analytical approach capable of measuring deaminated degradation products of oligonucleotide therapeutics is described. The method employs high-resolution mass spectrometry to assess the shift in isotopic distribution that accompanies deamination. Isotopic Distribution Factors (IDF), derived directly from the peak heights of the isotopic pattern, are employed to measure deamination levels of as little as 0.5%. Results obtained from application of the method to a phosphorothioate diester oligonucleotide exposed to various temperatures were used to determine deamination rates.

A new type of DNA phosphorothioation-based antiviral system in archaea.

Archaea and Bacteria have evolved different defence strategies that target virtually all steps of the viral life cycle. The diversified virion morphotypes and genome contents of archaeal viruses result in a highly complex array of archaea-virus interactions. However, our understanding of archaeal antiviral activities lags far behind our knowledges of those in bacteria. Here we report a new archaeal defence system that involves DndCDEA-specific DNA phosphorothioate (PT) modification and the PbeABCD-mediated halt of virus propagation via inhibition of DNA replication. In contrast to the breakage of invasive DNA by DndFGH in bacteria, DndCDEA-PbeABCD does not degrade or cleave viral DNA. The PbeABCD-mediated PT defence system is widespread and exhibits extensive interdomain and intradomain gene transfer events. Our results suggest that DndCDEA-PbeABCD is a new type of PT-based virus resistance system, expanding the known arsenal of defence systems as well as our understanding of host-virus interactions.