Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

The binding affinity-dependent inhibition of cell growth and viability by DNA sulfur-binding domains.

Increasing evidence suggests that DNA phosphorothioate (PT) modification serves several purposes in the bacterial host, and some restriction enzymes specifically target PT-DNA. PT-dependent restriction enzymes (PDREs) bind PT-DNA through their DNA sulfur binding domain (SBD) with dissociation constants (KD) of 5 nM~1 µM. Here, we report that SprMcrA, a PDRE, failed to dissociate from PT-DNA after cleavage due to high binding affinity, resulting in low DNA cleavage efficiency. Expression of SBDs in Escherichia coli cells with PT modification induced a drastic loss of cell viability at 25°C when both DNA strands of a PT site were bound, with one SBD on each DNA strand. However, at this temperature, SBD binding to only one PT DNA strand elicited a severe growth lag rather than lethality. This cell growth inhibition phenotype was alleviated by raising the growth temperature. An in vitro assay mimicking DNA replication and RNA transcription demonstrated that the bound SBD hindered the synthesis of new DNA and RNA when using PT-DNA as the template. Our findings suggest that DNA modification-targeting proteins might regulate cellular processes involved in DNA metabolism in addition to being components of restriction-modification systems and epigenetic readers.

Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

2',4'-LNA-Functionalized 5'-S-Phosphorothioester CDNs as STING Agonists.

Cyclic dinucleotides (CDNs) have garnered popularity over the last decade as immunotherapeutic agents, which activate the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway to trigger an immune response. Many analogs of 2'3'-cGAMP, c-di-GMP, and c-di-AMP have been developed and shown as effective cancer vaccines and immunomodulators for the induction of both the adaptive and innate immune systems. Unfortunately, the effectiveness of these CDNs is limited by their chemical and enzymatic instability. We recently introduced 5'-endo-phosphorothoiate 2'3'-cGAMP analogs as potent STING agonist with improved resistance to cleavage by clinically relevant phosphodiesterases. We herein report the synthesis of locked nucleic acid-functionalized (LNA) endo-S-CDNs and evaluate their ability to activate STING in THP1 monocytes. Interestingly, some of our synthesized LNA 3'3'-endo-S-CDNs can moderately activate hSTING REF haplotype (R232H), which exhibit diminished response to both 2'3'-cGAMP and ADU-S100. Also, we show that one of our most potent endo-S-CDNs has remarkable chemical (oxidants I2 and H2O2) and phosphodiesterase stability.

Controlling the Crystal Growth of DNA Molecules via Strategic Chemical Modifications.

Intermolecular interactions are critical to the crystallization of biomolecules, yet the precise control of biomolecular crystal growth based on these interactions remains elusive. To understand the connections between the crystallization kinetics and the strength of intermolecular interactions, herein we have employed DNA triangular crystals and modified ones as a versatile tool to investigate how the strength of intermolecular interaction affects crystal growth. Interestingly, we have found that the 2'-O-methylation at sticky ends of the DNA triangle could strengthen its intermolecular interaction, resulting in the accelerated formation of smaller crystals. Conversely, phosphorothioate modification could weaken the sticky-end cohesion and delay the nucleation, resulting in formation of fewer but larger crystals. In addition, these modification effects were consistently observed in the crystallization of a DNA decamer. In one word, our experimental results demonstrate that the strength of intermolecular interaction directly impacts crystal growth. It suggests that 2'-O-methylation and phosphorothioate modification represents a rational strategy for controlling DNA molecules grow into desired crystals and it also facilitates structural determination.

Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1.

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].

Harnessing sulfur-binding domains to separate Sp and Rp isomers of phosphorothioate oligonucleotides.

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60-90% of the starting Rp isomers, with PS linkage not locating at 5' or 3' end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method. KEY POINTS: • SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC. • SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers. • SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme.

Introns encode dsRNAs undetected by RIG-I/MDA5/interferons and sensed via RNase L.

Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase.

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3'-5' proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.

Inhibitory Effect of Phosphorothioate Oligonucleotide Complementary to G6PD mRNA on Murine Melanoma.

In terms of the incidence among all tumors, skin cancer is on top, with the most deadly among them being melanoma. The search for new therapeutic agents to combat melanoma is very relevant. In our opinion, antisense oligonucleotides (ASO) aimed at suppressing the genes responsible for their viability in cancer cells give hope for treatment, which makes it possible to eliminate cancer cells near the tumor site both before and after surgery. In this article, we describe how Skeen-11 phosphorothioate oligonucleotide significantly decreased the proliferative activity of murine melanoma cells. Injections of Skeen-11 also inhibited tumor growth in mice with inoculated melanoma. A toxicity study showed no side effects with dose adjustments. The results show that the use of ASO Skeen-11 in vivo reduced the tumor size within 7 days, reduced the number of mitoses in the tumor cells, and increased the amount of necrosis compared with the control group.

Alpha-thiol deoxynucleotide triphosphates (S-dNTPs) as radioprotective agents: A novel approach.

In this study, the ability of a mixture of four different alpha-thiol deoxynucleotide triphosphates (S-dNTPs) each at a concentration of 10µM when incorporated into the genomic DNA of proliferating human HL-60 and Mono-Mac-6 (MM-6) cells in vitro to provide protection from 2, 5, and 10 Gy of gamma radiation was investigated. Incorporation of the four different S-dNTPs into nuclear DNA at 10 µM concentration for five days was validated by agarose gel electrophoretic band shift analysis. S-dNTP-treated genomic DNA reacted with BODIPY-iodoacetamide demonstrated a band shift to higher molecular weight to confirm the presence of sulfur moieties in the resultant phosphorothioate DNA backbones. No overt signs of toxicity or obvious morphologic cellular differentiation were noted in the presence of 10 µM S-dNTPs even after 8 days in culture. Significantly reduced radiation-induced persistent DNA damage measured at 24 and 48 h post-exposure by γ-H2AX histone phosphorylation using FACS analysis in S-dNTP incorporated HL-60 and MM6 cells indicated protection against radiation-induced direct and indirect DNA damage. Statistically significant protection by S-dNTPs was noted at the cellular level by CellEvent™ Caspase-3/7 assay, which assess the extent of apoptotic events, and by trypan blue dye exclusion to assed cell viability. The results appear to support an innocuous antioxidant thiol radioprotective effect built into genomic DNA backbones as the last line of defense against ionizing radiation and free radical-induced DNA damage.

Discovery of Three Organothiophosphate Esters in River Water Using High-Resolution Mass Spectrometry.

Records of the environmental occurrence of organothiophosphate esters (OTPEs), which are used as flame retardants and food and industrial additives, are unavailable. In this study, we discovered three OTPEs, namely O,O,O-tris(2,4-di-tert-butylphenyl) phosphorothioate (AO168═S), O-butyl O-(butyl-methylphenyl) O-(di-butylphenyl) phosphorothioate (BBMDBPt)/O,O-bis(dibutylphenyl) O-methyl phosphorothioate (BDBPMPt), and O-butyl O-ethyl O-hydrogen phosphorothioate (BEHPt), in the surface water of the Yangtze River Basin by applying a characteristic phosphorothioate fragment-directed high-resolution mass spectrometry method. Among the 17 water samples tested, the detection frequencies of AO168═S and BEHPt were 100% and that of BBMDBPt/BDBPMPt was 29%. The mean concentration of AO168═S was 56.9 ng/L (30.5-148 ng/L), and semi-quantitative analysis revealed that the mean concentrations of BEHPt and BBMDBPt/BDBPMPt were 17.2 ng/L (5.5-65.4 ng/L) and 0.8 ng/L (

From CPG to hybrid support: Review on the approaches in nucleic acids synthesis in various media.

Solid-phase synthesis is, to date, the preferred method for the manufacture of oligonucleotides, in quantities ranging from a few micrograms for research purposes to several kilograms for therapeutic or commercial use. But for large-scale oligonucleotide manufacture, scaling up and hazardous waste production pose challenges that necessitate the investigation of alternate synthetic techniques. Despite the disadvantages of glass supports, using soluble supports as a substitute presents difficulties because of their high overall yield and complex purification steps. To address these challenges, various independent approaches have been developed; however, other problems such as insufficient cycle efficiency and synthesis of oligonucleotide chains of desired length continue to exist. In this study, we present a review of the current developments, advantages, and difficulties of recently reported alternatives to supports based on controlled pore glass, and discuss the importance of a support choice to resolve issues arising during oligonucleotide synthesis.

Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.

Ensemble Modified Aptamer Based Pattern Recognition for Adaptive Target Identification.

The difficulty of the molecular design and chemical synthesis of artificial sensing receptors restricts their diagnostic and proteomic applications. Herein, we report a concept of "ensemble modified aptamers" (EMAmers) that exploits the collective recognition abilities of a small set of protein-like side-chain-modified nucleic acid ligands for discriminative identification of molecular or cellular targets. Different types and numbers of hydrophobic functional groups were incorporated at designated positions on nucleic acid scaffolds to mimic amino acid side chains. We successfully assayed 18 EMAmer probes with differential binding affinities to seven proteins. We constructed an EMAmer-based chemical nose sensor and demonstrated its application in blinded unknown protein identification, giving a 92.9% accuracy. Additionally, the sensor is generalizable to the detection of blinded unknown bacterial and cellular samples, which enabled identification accuracies of 96.3% and 94.8%, respectively. This sensing platform offers a discriminative means for adaptive target identification and holds great potential for diverse applications.

Photoinduced Synthesis of Sulfonyl-Containing Phosphorothioates via a Three-Component Reaction.

Both sulfonyl and phosphorothioate are important privileged structural motifs which are widely presented in pharmaceuticals and agrochemicals. Herein, we describe an efficient approach to synthesizing sulfonyl-containing phosphorothioates by merging photoredox and copper catalysis at room temperature. This protocol is compatible with a wide range of substrates and can be applied to the late-stage modification of complex molecules. Control experiments are conducted to demonstrate the generation of the sulfonyl radical in the transformation.

Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties.

Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti-miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents.

Antisense Oligonucleotides Conjugated with Lipophilic Compounds: Synthesis and In Vitro Evaluation of Exon Skipping in Duchenne Muscular Dystrophy.

Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (Tm) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.