Phosphorothioate Research Articles

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.

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers.

Phosphorothioate (PS) modification of antisense oligonucleotides (ASOs) is a critical factor enabling their therapeutic use. Standard chemical synthesis incorporates this group in a stereorandom manner; however, significant effort was made over the years to establish and characterize the impact of chiral control. In this work, we present our in-depth characterization of interactions between Escherichia coli RNase H and RNA-DNA heteroduplexes carrying chirally defined PS groups. First, using a massive parallel assay, we showed that at least a single Rp-PS group is necessary for efficient RNase H-mediated cleavage. We followed by demonstrating that this group needs to be aligned to the phosphate-binding pocket of RNase H, and that chiral status of other PS groups in close proximity to RNase H does not affect cleavage efficiency. We have shown that RNase H's PS chiral preference can be utilized to guide cleavage to a specific chemical bond. Finally, we present a strategy for ASO optimization by mapping preferred RNase H cleavage sites of a non-thioated compound, followed by introduction of Rp-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an Sp-PS at the same location.

Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses.

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT‐containing diribonucleotides with native species in RNA hydrolysates by high‐resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT‐specific iodine‐desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′‐O‐methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.

Efficient Synthesis of 2'-O-Methoxyethyl Oligonucleotide-Cationic Peptide Conjugates.

Single‐stranded phosphorothioate (PS) oligonucleotide drugs have shown potential for the treatment of several rare diseases. However, a barrier to their widespread use is that they exhibit activity in only a narrow range of tissues. One way to circumvent this constraint is to conjugate them to cationic cell‐penetrating peptides (CPPs). Although there are several examples of morpholino and peptide nucleic acids conjugated with CPPs, there are noticeably few examples of PS oligonucleotide‐CPP conjugates. This is surprising given that PS oligonucleotides presently represent the largest class of approved RNA‐based drugs, including Nusinersen, that bears the 2’‐O‐methoxyethyl (MOE)‐chemistry. In this work, we report a method for in‐solution conjugation of cationic, hydrophobic peptides or human serum albumin to a 22‐nucleotide MOE‐PS oligonucleotide. Conjugates were obtained in high yields and purities. Our findings pave the way for their large‐scale synthesis and testing in vivo.

Synthesis, Stability, and Anti-Tumour Activity of a New Category of "Stapled" Antisense Oligonucleotides with Stimuli-Responsive Feature.

The development of nucleic acid drugs with unique structures and mechanisms has stimulated great research interest. Herein, we report a general strategy to construct "stapled" structures of single-stranded antisense oligonucleotides (ASONs) with a stimuli-responsive feature. "Stapled" cyclic structures can be synthesized with reactive bifunctional handles that react with thiol groups of phosphorothioate (PS)-modified ASONs, and can be alternatively adjusted depending on the desired PS sites in the ASON strand. The disulphide group in the stapled handle can be cleaved in the reducing microenvironment of tumour cells. Thus, "stapled" ASONs may be transformed back to a linear conformation to facilitate binding to target mRNAs. Stapling conferred protection against degradation, and enhanced anti-tumour activity compared to linear counterparts. This study provides a new, effective, and convenient strategy for designing ASONs with "stapled" structures, and also adds a further contribution to facilitate the stability and biological efficacy of novel nucleic acid-based therapeutics.

Site-Specific Scissors Based on Myeloperoxidase for Phosphorothioate DNA.

The tool box of site-specific cleavage for nucleic acid has been an increasingly attractive subject. Especially, the recent emergence of the orthogonally activatable DNA device is closely related to the site-specific scission. However, most of these cleavage strategies are based on exogenous assistance, such as laser irradiation. Endogenous strategies are highly desirable for the orthogonally regulatable DNA machine to explore the crucial intracellular biological process and cell signal network. Here, we found that the accurate site-specific cleavage reaction of phosphorothioate (PT) modified DNA by using myeloperoxidase (MPO). A scissors-like mechanism by which MPO breaks PT modification through chloride oxidation has been revealed. Furthermore, we have successfully applied the scissors to activate PT-modified hairpin-DNA machines to produce horseradish peroxidase (HRP)-mimicking DNAzyme or initiate hybridization chain reaction (HCR) amplification. Since MPO plays an important role in the pathway related to oxidative stress in cells, through the HCR amplification activated by this tool box, the oxidative stress in living cells has been robustly imaged. This work proposes an accurate and endogenous site-specific cleavage tool for the research of biostimuli and the construction of DNA molecular devices.

Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo.

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5′ end and Sp diastereomers at the 3′ end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5′ end and Sp isomer at the 3′ end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.

Selective Anti-melanoma Effect of Phosphothioated Aptamer Encapsulated by Neutral Cytidinyl/Cationic Lipids.

BC15-31 is a DNA aptamer that targets heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which plays a crucial role in the process of pre-RNA maturation and is also essential for the rapid proliferation of tumor cells. In this research, we modified BC15-31 with a phosphorothioate (PS) backbone, LNA, and 2-O-MOE to enhance its stability and target affinity. In addition, a neutral cytidinyl lipid (DNCA) and a cationic lipid (CLD) were mixed to encapsulate modified aptamers with the aim of improving their cell permeability with low toxicity. Under the DNCA/CLD package, aptamers are mainly distributed in the nucleus. A modified sequence WW-24 showed an excellent selective anti-melanoma (A375 cells, ∼25 nM, 80%) activity, targeted to both hnRNP A1 and hnRNP A2/B1 found by the BLI experiment, and induced more early and late apoptosis in vitro, which also showed stronger antitumor effect and longer accumulation time in vivo. These results provide a new strategy for further clinical applications.

Nucleophilic Substitution at Tetracoordinate Phosphorus. Stereochemical Course and Mechanisms of Nucleophilic Displacement Reactions at Phosphorus in Diastereomeric cis- and trans-2-Halogeno-4-methyl-1,3,2-dioxaphosphorinan-2-thiones: Experimental and DFT Studies.

Geometrical cis- and trans- isomers of 2-chloro-, 2-bromo- and 2-fluoro-4-methyl-1,3,2-dioxaphosphorinan-2-thiones were obtained in a diastereoselective way by (a) sulfurization of corresponding cyclic PIII-halogenides, (b) reaction of cyclic phosphorothioic acids with phosphorus pentachloride and (c) halogen–halogen exchange at PIV-halogenide. Their conformation and configuration at the C4-ring carbon and phosphorus stereocentres were studied by NMR (1H, 31P) methods, X-ray analysis and density functional (DFT) calculations. The stereochemistry of displacement reactions (alkaline hydrolysis, methanolysis, aminolysis) at phosphorus and its mechanism were shown to depend on the nature of halogen. Cyclic cis- and trans-isomers of chlorides and bromides react with nucleophiles (HO−, CH3O−, Me2NH) with inversion of configuration at phosphorus. DFT calculations provided evidence that alkaline hydrolysis of cyclic thiophosphoryl chlorides proceeds according to the SN2-P mechanism with a single transition state according to the potential energy surface (PES) observed. The alkaline hydrolysis reaction of cis- and trans-fluorides afforded the same mixture of the corresponding cyclic thiophosphoric acids with the thermodynamically more stable major product. Similar DFT calculations revealed that substitution at phosphorus in fluorides proceeds stepwise according to the A–E mechanism with formation of a pentacoordinate intermediate since a PES with two transition states was observed.

SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System.

Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.IMPORTANCE We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system. To our surprise, ssDNA PT modification can also pair with a newly identified 3-gene sspFGH cassette to fend off phage infection with a different mode of action from that of SspE. Interestingly, both SspFGH and SspE can pair with the same SspABCD module for antiphage defense, and their combination provides Escherichia coli JM109 with additive phage resistance up to 105-fold compared to that for either barrier alone. This agrees with our observation that SspFGH and SspE coexist in 36 bacterial genomes, highlighting the diversity of the gene contents and molecular mechanisms of PT-based defense systems.

Nanoparticle Delivered Anti-miR-141-3p for Stroke Therapy.

Ischemic stroke and factors modifying ischemic stroke responses, such as social isolation, contribute to long-term disability worldwide. Several studies demonstrated that the aberrant levels of microRNAs contribute to ischemic stroke injury. In prior studies, we established that miR-141-3p increases after ischemic stroke and post-stroke isolation. Herein, we explored two different anti-miR oligonucleotides; peptide nucleic acid (PNAs) and phosphorothioates (PS) for ischemic stroke therapy. We used US FDA approved biocompatible poly (lactic-co-glycolic acid) (PLGA)-based nanoparticle formulations for delivery. The PNA and PS anti-miRs were encapsulated in PLGA nanoparticles by double emulsion solvent evaporation technique. All the formulated nanoparticles showed uniform morphology, size, distribution, and surface charge density. Nanoparticles also exhibited a controlled nucleic acid release profile for 48 h. Further, we performed in vivo studies in the mouse model of ischemic stroke. Ischemic stroke was induced by transient (60 min) occlusion of middle cerebral artery occlusion followed by a reperfusion for 48 or 72 h. We assessed the blood-brain barrier permeability of PLGA NPs containing fluorophore (TAMRA) anti-miR probe after systemic delivery. Confocal imaging shows uptake of fluorophore tagged anti-miR in the brain parenchyma. Next, we evaluated the therapeutic efficacy after systemic delivery of nanoparticles containing PNA and PS anti-miR-141-3p in mice after stroke. Post-treatment differentially reduced both miR-141-3p levels in brain tissue and infarct injury. We noted PNA-based anti-miR showed superior efficacy compared to PS-based anti-miR. Herein, we successfully established that nanoparticles encapsulating PNA or PS-based anti-miRs-141-3p probes could be used as a potential treatment for ischemic stroke.

Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3′ end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3′ end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.

Site-specific Incorporation of 2',5'-Linked Nucleic Acids Enhances Therapeutic Profile of Antisense Oligonucleotides.

Site-specific incorporation of 2'-modifications and neutral linkages in the deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined the effect of introducing 2',5'-linked RNA in the deoxynucleotide gap region on toxicity and potency of PS ASOs. Our results demonstrate that incorporation of 2',5'-linked RNA in the gap region dramatically improved hepatotoxicity profile of PS-ASOs without compromising potency and provide a novel alternate chemical approach for improving therapeutic index of ASO drugs.

Single-molecule optical mapping of the distribution of DNA phosphorothioate epigenetics

DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.

Abstract P774: Therapeutic Efficacy of Next Generation Anti-miR-141-3p for Ischemic Stroke

Background: Stroke and factors modifying stroke responses, such as social isolation, increases miR-141-3p. Hence, inhibiting miR-141-3p can be a promising stroke therapy. Unfortunately, commercially available miR-141-3p inhibitors cannot be used for therapeutic purpose due to several limitations; use of viral or non-viral transfection reagents for cell penetration, in vivo instability of anti-miRs and toxicity of synthetic anti-miRs. Therefore, we explored phosphorothioates (PS) and peptide nucleic acids (PNAs) based anti-miR-141-3p encapsulated in poly (lactide-co-glycolide) (PLGA)-based nanoparticles (NPs) for potential stroke therapy. Methods: PNAs and PS anti-miR 141-3p were synthesized in house using solid phase synthesis and purchased from a commercial vendor, respectively. The anti-miRs were encapsulated in PLGA NP by double emulsion solvent evaporation technique. For in vivo efficacy 8-10 weeks old C57BL/6 male mice were pair-housed (PH) for at least two weeks. After two weeks, a 60-minute right middle cerebral artery occlusion surgery (MCAO) was performed and mice were kept individually. The mice were then randomly assigned to receive NPs of either anti-miR-141-3p PNA or anti-miR-141-3p phosphorothioate (PS) or scrambled control (Sc) through lateral tail vein 4 hrs. after stroke. To measure NPs of PS were conjugated with fluorophore (TAMRA) to measure BBB permeability. The effect of inhibitor treatment was evaluated at 3 days after stroke. Results: The formulated NPs show uniform morphology with a mean dry particle size <120 nm and an average hydrodynamic diameter of 350 nm. The NPs exhibit an average polydispersity index of 0.1-0.2 indicating homogenous particle size distribution. Single dose of anti-miR 141 and (NPs containing 0.05mg/kg i.v of total either PNA or PS) Both the NPs of PNA and PS significantly reduced (P<0.05 vs. Sc) infarct injury and neurological deficit. Anti-miR of PNA and PS reduce 6 and 5-fold respectively of miR-141-3p in the brain tissue. Conclusion: Our NPs are BBB permeable. PNA based anti-miR-141 are more potent the PS based anti-miR Reduced miR-141-3p levels as well as infarct injury by both PS and PNA based anti-miR 141-3p suggest the efficacy of our novel ant-miR -141-3p for the treatment of ischemic stroke.

History of Research on Antisense Oligonucleotide Analogs

In the search for novel therapeutics, antisense oligonucleotide (ASO) analogs have been a major focus of research for over 40 years. They use the antisense strategy, namely they have a nucleic acid base sequence that is complementary to a portion of a specific mRNA that is produced in the cell, or to a viral RNA, in order to selectively inhibit gene expression. Oligonucleotides need to be chemically modified to stabilize them against hydrolysis by endogenous nucleases. Until now several phosphorothioate (PS) oligonucleotide analogs have been approved by the FDA for human use. This article seeks to provide a history of this subject to date.

Generation of αCD11b-CpG antibody conjugates for the targeted stimulation of myeloid cells

CpG oligonucleotides are short single-stranded synthetic DNA molecules. Upon binding to Toll-like receptor 9 (TLR9), CpG activates immune cells in humans and mice. This results in robust Th1 type immunity potentially resulting in clearance of pathogens, reduction of allergy and anti-tumor immunity. However, the effectiveness of CpG as an adjuvant depends on its administration route, with only strong effects seen when CpG is administered locally. As local administration is not always feasible, we generated conjugates to specifically deliver CpG to myeloid cells often abundantly present in tumors. For this we coupled CpG (3′-Thiol-modified phosphorothioate (PTO) CpG-ODN1826 type B (5′-tccatgacgttcctgacgtt-3′)) to monoclonal antibodies (mAbs) directed against the myeloid cell marker CD11b using maleimide-thiol coupling. The CD11b-CpG mAb (αCD11b-CpG) conjugates contained about four CpG molecules/conjugate and displayed binding and internalization characteristics similar to unconjugated CD11b mAbs (αCD11b). The αCD11b-CpG conjugates readily induced maturation of murine dendritic cells (DCs) in a TLR9-dependent manner in vitro. Following intravenous injection, αCD11b-CpG conjugates efficiently targeted CD11b+ immune cells in the blood, lymph nodes and spleen. Finally, injection of αCD11b-CpG conjugates, but not untargeted conjugates, induced maturation of CD11b+ cell subsets in vivo. In conclusion, conjugating CpG to αCD11b enabled specific targeting and activation of myeloid cells in vivo.

Site-specific incorporation of 5′-methyl DNA enhances the therapeutic profile of gapmer ASOs

We recently showed that site-specific incorporation of 2′-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5′-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5′-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2′-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5′-Me and R-5′-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5′-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.

Self-priming phosphorothioated hairpin-mediated isothermal amplification

We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection. Isothermal amplification strategies are a simple process that efficiently raises the amount of nucleic acid at a constant temperature, but still has lots of problems such as the requirement of multiple exogenous primers and enzymes, which trigger non-specific background signal and increase the complexity of procedures. The key component for overcoming the above-mentioned limitations is the designed hairpin probe (HP) consisting of self-priming region along the 3′ stem and the 3′ overhang and phosphorothioate modifications at the 5′ overhang and the specific loop part. The HP was designed to open through binding to target nucleic acid. Upon opening of HP, its self-priming (SP) region is rearranged to form a smaller hairpin whose 3′ end could serve as a primer. The following extension produces the extended HP and displaces the bound target nucleic acid, which is then recycled to open another HP. Due to the reduced stability caused by the specific two phosphorothioate (PS) modifications, the 3′ end of EP1 is readily rearranged to form the foldback hairpin structure, which would promote the foldback extension to produce once more extended HP. Since the two PS modifications are always located at the same positions along the 5’ stem within the further extended HPs, the foldback reaction followed by the extension would be continuously repeated, consequently producing a large number of the long hairpin concatamers. Based on this unique design principle, we successfully detected even a single copy of target DNA with outstanding discrimination capability under an isothermal condition by employing only a single HP without the requirement for the complicated multiple primers. In conclusion, the sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling short target nucleic acid detection such as microRNAs or any target which is less than 200 mer.