Advances in structural-guided modifications of siRNA.

To date, the US Food and Drug Administration (FDA) has approved six small interfering RNA (siRNA) drugs: patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran, serving as compelling evidence of the promising potential of RNA interference (RNAi) therapeutics. The successful implementation of siRNA therapeutics is improved through a combination of various chemical modifications and diverse delivery approaches. The utilization of chemically modified siRNA at specific sites on either the sense strand (SS) or antisense strand (AS) has the potential to enhance resistance to ribozyme degradation, improve stability and specificity, and prolong the efficacy of drugs. Herein, we provide comprehensive analyses concerning the correlation between chemical modifications and structure-guided siRNA design. Various modifications, such as 2'-modifications, 2',4'-dual modifications, non-canonical sugar modifications, and phosphonate mimics, are crucial for the activity of siRNA. We also emphasize the essential strategies for enhancing overhang stability, improving RISC loading efficacy and strand selection, reducing off-target effects, and discussing the future of targeted delivery.

MicroLOCK: Highly stable microgel biosensor using locked nucleic acids as bioreceptors for sensitive and selective detection of let-7a.

Chemically modified oligonucleotides can solve biosensing issues for the development of capture probes, antisense, CRISPR/Cas, and siRNA, by enhancing their duplex-forming ability, their stability against enzymatic degradation, and their specificity for targets with high sequence similarity as microRNA families. However, the use of modified oligonucleotides such as locked nucleic acids (LNA) for biosensors is still limited by hurdles in design and from performances on the material interface. Here we developed a fluorogenic biosensor for non-coding RNAs, represented by polymeric PEG microgels conjugated with molecular beacons (MB) modified with locked nucleic acids (MicroLOCK). By 3D modeling and computational analysis, we designed molecular beacons (MB) inserting spot-on LNAs for high specificity among targets with high sequence similarity (95%). MicroLOCK can reversibly detect microRNA targets in a tiny amount of biological sample (2 µL) at 25 °C with a higher sensitivity (LOD 1.3 fM) without any reverse transcription or amplification. MicroLOCK can hybridize the target with fast kinetic (about 30 min), high duplex stability without interferences from the polymer interface, showing high signal-to-noise ratio (up to S/N = 7.3). MicroLOCK also demonstrated excellent resistance to highly nuclease-rich environments, in real samples. These findings represent a great breakthrough for using the LNA in developing low-cost biosensing approaches and can be applied not only for nucleic acids and protein detection but also for real-time imaging and quantitative assessment of gene targeting both in vitro and in vivo.

Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification.

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3' end of the sense strand of siRNA and cholesterol on the silencing activity of "light" and "heavy" modified siRNAs. All 3'-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5'-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3'-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

Aptasensor-encapsulating semi-permeable proteinosomes for direct target detection in non-treated biofluids.

Detecting biomarkers in biofluids directly without sample treatments makes molecular diagnostics faster and more efficient. Aptasensors, the nucleic acid-based molecular biosensors, can detect a wide range of target molecules, but their susceptibility to degradation and aggregation by nucleases and charged proteins, respectively, limits their direct use in clinical samples. In this work, we demonstrate that when aptasensors are encapsulated in proteinosomes, the protein-based liposome mimics, clinically important small molecules can be sensitively and selectively detected in non-treated specimens, such as 100 % unpurified serum. As serum albumin is used to form the membrane, the nanomeshed proteinosomes become semi-permeable and antifouling, which enables exclusive admission of small molecules while blocking unwanted large proteins. Consequently, the enclosed aptasensors can maintain close-to-optimal performance for target binding, and nucleolytic degradation and electrostatic aggregation are effectively suppressed. Three different structure-switching aptamers specific for estradiol, dopamine, and cocaine, respectively, are demonstrated to fully conserve their high affinities and specificities inside the microcapsules. The shielding effect of proteinosomes is indeed exceptional; the enclosed DNA aptasensors remain completely intact over 18 h in serum and even in an extremely concentrated DNase solution (1 mg/ml, ∼300,000× the serum level). Moreover, the proteinosome-mediated compartmentalization enables independent operation of multiple aptasensors in the same mixture. Hence, simultaneous real-time sensing of two different targets is demonstrated with different operation modes, 'recording' target appearance and 'reporting' target concentration changes. This work is the first demonstration of small-molecule-specific aptasensors operating with optimal performance in serum environments and will find promising applications in molecular diagnostics.

Enzymatic Synthesis of TNA Protects DNA Nanostructures.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3' terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

N3-Methyluridine and 2'-O-Alkyl/2'-Fluoro-N3-methyluridine functionalized nucleic acids improve nuclease resistance while maintaining duplex geometry.

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-m3U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT20 with 2'-O-alkyl-m3U modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to m3U, 2'-F, 2'-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m3U and 2'-O-methyl-m3U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2'-O-propyl-m3U modification exhibits steric interactions with amino acid residues in the active site of 3'- and 5'-exonuclease, leading to enhanced stability. These combined data indicate that the 2'-modified-m3U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics.

Topology- and size-dependent binding of DNA nanostructures to the DNase I.

The susceptibility of DNA nanomaterials to enzymatic degradation in biological environments is a significant obstacle limiting their broad applications in biomedicine. While DNA nanostructures exhibit some resistance to nuclease degradation, the underlying mechanism of this resistance remains elusive. In this study, the interaction of tetrahedral DNA nanostructures (TDNs) and double-stranded DNA (dsDNA) with DNase I is investigated using all-atom molecular dynamics simulations. Our results indicate that DNase I can effectively bind to all dsDNA molecules, and certain key residues strongly interact with the nucleic bases of DNA. However, the binding of DNase I to TDNs exhibits a non-monotonic behavior based on size; TDN15 and TDN26 interact weakly with DNase I (∼ - 75 kcal/mol), whereas TDN21 forms a strong binding with DNase I (∼ - 110 kcal/mol). Furthermore, the topological properties of the DNA nanostructures are analyzed, and an under-twisting (∼32°) of the DNA helix is observed in TDN15 and TDN26. Importantly, this under-twisting results in an increased width of the minor groove in TDN15 and TDN26, which primarily explains their reduced binding affinity to DNase I comparing to the dsDNA. Overall, this study demonstrated a novel mechanism for local structural control of DNA at the nanoscale by adjusting the twisting induced by length.

Protonation-mediated DNA tile self-assembly with nuclease resistance characteristic for signal-amplified detection of microRNAs.

DNA nanotechnology, developing rapidly in recent years, has unprecedented superiorities in biological application-oriented research including high programmability, convenient functionalization, reconfigurable structure, and intrinsic biocompatibility. However, the susceptibility to nucleases in the physiological environment has been an obstacle to applying DNA nanostructures in biological science research. In this study, a new DNA self-assembly strategy, mediated by double-protonated small molecules instead of classical metal ions, is developed to enhance the nuclease resistance of DNA nanostructures while retaining their integrality and functionality, and the relative application has been launched in the detection of microRNAs (miRNAs). Faced with low-abundance miRNAs, we integrate hybrid chain reaction (HCR) with DNA self-assembly in the presence of double-protonated small molecules to construct a chemiluminescence detection platform with nuclease resistance, which utilizes the significant difference of molecular weight between DNA arrays and false-positive products to effectively separate of reaction products and remove the detection background. This strategy attaches importance to the nucleic acid stability during the assay process via improving nuclease resistance while rendering the detection results for miRNAs more authentic and reliable, opening our eyes to more possibilities for the multiple applications of customized DNA nanostructures in biology, including bioassay, bioimaging, drug delivery, and cell modulation.

Selection, activity, and nuclease stabilization of cross-neutralizing DNA aptamers targeting HSV-1 and HSV-2.

Cross-neutralizing aptamers targeting both HSV-1 and HSV-2 were developed by selecting against the ectodomains of glycoprotein D (gD) from both viruses in parallel as well as sequentially using the SELEX method. Since gD facilitates viral invasion, sterically blocking the host-receptor interaction prevents infection. Candidate aptamers were screened, and lead aptamers were identified that exhibited exceptional neutralizing activity against both viruses in vitro. The specificity of the aptamers was confirmed by comparing their activity to scrambled versions of themselves. Modifications of the lead compounds were tested to define critical motifs to guide development. Stability of the aptamers was increased using phosphorothioate backbone linkages, and 2' methoxy substitutions of terminal and key internal bases. Aptamers were applied in a guinea pig vaginal HSV-2 infection model and found to reduce both the viral load of infected animals and the severity of the resulting disease. These results suggest that cross-neutralizing aptamers can be developed into on-demand antiviral interventions effective against both HSV-1 and HSV-2.

Construction of a 3D rigidified DNA nanodevice for anti-interference and reinforced biosensing by turning nuclease into a catalyst.

The practical application of DNA biosensors is impeded by numerous limitations in complicated physiological environments, particularly the susceptibility of common DNA components to nuclease degradation, which has been recognized as a major barrier in DNA nanotechnology. In contrast, the present study presents an anti-interference and reinforced biosensing strategy based on a 3D DNA-rigidified nanodevice (3D RND) by converting a nuclease into a catalyst. 3D RND is a well-known tetrahedral DNA scaffold containing four faces, four vertices, and six double-stranded edges. The scaffold was rebuilt to serve as a biosensor by embedding a recognition region and two palindromic tails on one edge. In the absence of a target, the rigidified nanodevice exhibited enhanced nuclease resistance, resulting in a low false-positive signal. 3D RNDs have been proven to be compatible with 10% serum for at least 8 h. Once exposed to the target miRNA, the system can be unlocked and converted into common DNAs from a high-defense state, followed by polymerase- and nuclease-co-driven conformational downgrading to achieve amplified and reinforced biosensing. The signal response can be improved by approximately 700% within 2 h at room temperature, and the limit of detection (LOD) is approximately 10-fold lower under biomimetic conditions. The final application to serum miRNA-mediated clinical diagnosis of colorectal cancer (CRC) patients revealed that 3D RND is a reliable approach to collecting clinical information for differentiating patients from healthy individuals. This study provides novel insights into the development of anti-interference and reinforced DNA biosensors.

Synthesis and characterization of novel (S)-5'-C-aminopropyl-2'-fluorouridine modified oligonucleotides as therapeutic siRNAs.

The lack of stability of natural nucleosides limits their application in small interfering RNA (siRNA)-mediated RNA interference (RNAi). Various chemical modifications have been reported to improve their pharmacokinetic behavior; however, the development of potential candidates is still underway. In this study, we designed and synthesized (S)-5'-C-aminopropyl-2'-fluorouridine (5'-AP-2'-FU) and evaluated the properties of siRNAs containing this analog. A comparative thermodynamic study revealed the enhanced thermal stability of double-stranded RNAs (dsRNAs) containing 5'-AP-2'-FU in a position-specific manner, whereas (S)-5'-C-aminopropyl-2'-O-methyluridine (5'-AP-2'-MoU)-modified dsRNAs exhibited lower melting temperatures. This improved thermal stability of RNA duplexes is attributed to favorable entropy loss, which induces the duplex into an N-type (C3'-endo) conformation and enhances duplex　binding in this case. The 5'-AP-2'-FU analog was also suitable for incorporation into the passenger strand to induce gene-silencing activity. Gene knockdown efficacy was comparable to that of unmodified siRNAs, and the best response was observed by introducing 5'-AP-2'-FU near the 3'-terminal end of the passenger strand. In addition, the single-stranded RNAs (ssRNAs) modified with 5'-AP-2'-FU showed strong resistance against decomposition by nucleases when treated with buffer containing bovine serum, which was similar to 5'-AP-2'-MoU.

Synthesis of oligonucleotides containing 5'-homo-4'-selenouridine derivative and its increased resistance against nuclease.

As technologies using RNA or DNA have been developed, various chemical modifications of nucleosides have been attempted to increase the stability of oligonucleotides. Since it is known that 2'-OMe-modification greatly contributes to increasing the stability of oligonucleotides, we added 2'-OMe to our previously developed 4'-selenonucleoside and 5'-homo-4'-selenonucleoside as the modified monomers for oligonucleotide: 2'-methoxy-4'-selenouridine (2'-OMe-4'-Se-U) and 5'-homo-2'-methoxy-4'-selenouridine (5'-homo-2'-OMe-4'-Se-U). We synthesized oligonucleotides containing the chemically modified 4'-selenouridine and evaluated their thermal stability and nuclease resistance. In conclusion, the nuclease stability of the oligonucleotide containing 5'-homo-2'-OMe-4'-selenouridine increased while its thermal stability decreased.

bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements.

Monoclonal antibody therapies targeting immuno-modulatory targets such as checkpoint proteins, chemokines, and cytokines have made significant impact in several areas, including cancer, inflammatory disease, and infection. However, antibodies are complex biologics with well-known limitations, including high cost for development and production, immunogenicity, a limited shelf-life because of aggregation, denaturation, and fragmentation of the large protein. Drug modalities such as peptides and nucleic acid aptamers showing high-affinity and highly selective interaction with the target protein have been proposed alternatives to therapeutic antibodies. The fundamental limitation of short in vivo half-life has prevented the wide acceptance of these alternatives. Covalent drugs, also known as targeted covalent inhibitors (TCIs), form permanent bonds to target proteins and, in theory, eternally exert the drug action, circumventing the pharmacokinetic limitation of other antibody alternatives. The TCI drug platform, too, has been slow in gaining acceptance because of its potential prolonged side-effect from off-target covalent binding. To avoid the potential risks of irreversible adverse drug effects from off-target conjugation, the TCI modality is broadening from the conventional small molecules to larger biomolecules possessing desirable properties (e.g., hydrolysis resistance, drug-action reversal, unique pharmacokinetics, stringent target specificity, and inhibition of protein-protein interactions). Here, we review the historical development of the TCI made of bio-oligomers/polymers (i.e., peptide-, protein-, or nucleic-acid-type) obtained by rational design and combinatorial screening. The structural optimization of the reactive warheads and incorporation into the targeted biomolecules enabling a highly selective covalent interaction between the TCI and the target protein is discussed. Through this review, we hope to highlight the middle to macro-molecular TCI platform as a realistic replacement for the antibody.

In situ imaging miRNAs using multifunctional linear DNA nanostructure.

The microRNAs (miRNAs) play a critical role in many biological processes and are essential biomarkers for diagnosing disease. However, the sensitive and specific quantification of microRNAs (miRNAs) expression in living cells still faces a huge challenge. Our study designed a multifunctional linear DNA nanostructure (MLN) as a carrier of molecular beacons (MB-21) for detecting and intracellular imaging miRNA-21. The MLN-MB consists of three parts: aptamer, MLN, and MB-21. The aptamer (AS1411) could media MLN-MB enter live cells without additional transfection reagents. Once inside the cells, the intracellular miRNA-21 could hybridize the MB-21s, resulting in significantly enhanced fluorescence signals. The whole process was enzyme-free, autonomous, and continuous, which avoided the necessity of adding external fuel strands or enzymes. We demonstrated that the MLN-MB could be used to screen the miRNA-21 with a detection limit of 320 pM in a short time (10 min) and show high specificity toward miRNA-21 against other miRNAs. Moreover, the proposed MLN-MB could detect the miRNA-21 in complex matrixes stably. With its outstanding stability, dual recognition, and biocompatibility, MLN-MB is capable of delivering into living cells to identify specific cancer cells. Therefore, our sensing approach, with high sensitivity, specificity, and simplicity advantages, holds great potential for early cancer diagnosis.

Synthesis and properties of fully-modified 4'-selenoRNA, an endonuclease-resistant RNA analog.

A large number of chemically modified oligonucleotides (ONs) have been developed for RNA-based technologies. In most modified RNAs, the characteristic 2'-hydroxyl (2'-OH) groups are removed to enhance both nuclease resistance and hybridization ability. However, the importance of the 2'-OH group in RNA structure and function is well known. Here, we report the synthesis and properties of 4'-selenoRNA in which all four nucleoside units retain the 2'-OH groups but contain a selenium atom instead of an oxygen atom at the 4'-position of the furanose ring. 4'-SelenoRNA has enhanced ability to form duplexes with RNA, and high endonuclease resistance despite the presence of the 2'-OH groups. X-ray crystallography analysis showed that the 4'-selenoRNA duplex adopts an A-conformation, similar to natural RNA, although one 4'-selenocytidine residue has unusual South-type sugar puckering. Furthermore, preliminary studies using 4'-seleno-modified siRNAs suggest that 4'-selenoRNA may be applicable to RNA interference technology. Collectively, our results raise the possibility of a new class of modified RNA in which 2'-OH groups do not need to be masked.

Nuclease resistance and protein recognition properties of DNA and hybrid PNA-DNA four-way junctions.

Nucleic acids possess unique biochemical features that make them ideal candidates to inhibit "difficult to target" proteins. The limited stability of nucleic acids in vivo presents a major obstacle to their development as drugs. Here, immobile four-way junctions (4WJs) are used to target the DNA-binding cytokine, High Mobility Group B1. Hybrid 4WJs composed of DNA and peptide nucleic acids (PNA) are investigated. PNA possess enhanced nuclease stability vs. DNA. 4WJs are incubated with Exonuclease III and DNase I. The nuclease assays show that 4WJs containing multiple PNAs possess significantly higher stability. Circular dichroism assays are used to probe the groove topology of 4WJs with the minor groove binder, DAPI. The CD data indicates that multi-PNA 4WJs possess altered minor groove dimensions that reduces DAPI binding affinity. Logic suggests that the minor groove of multi-PNA hybrids possess significant perturbations to the topology and local electrostatic environment that prevents proper binding/recognition by nucleases and thus enhances stability.

Relative Nuclease Resistance of a DNA Aptamer Covalently Conjugated to a Target Protein.

A major obstacle to the therapeutic application of an aptamer is its susceptibility to nuclease digestion. Here, we confirmed the acquisition of relative nuclease resistance of a DNA-type thrombin binding aptamer with a warhead (TBA3) by covalent binding to a target protein in the presence of serum/various nucleases. When the thrombin-inhibitory activity of TBA3 on thrombin was reversed by the addition of the complementary strand, the aptamer was instantly degraded by the nucleases, showing that the properly folded/bound aptamer conferred the resistance. Covalently binding aptamers possessing both a prolonged drug effect and relative nuclease resistance would be beneficial for in vivo translational applications.

4'-C-Aminoethoxy-Modified DNAs Exhibit Increased Nuclease Resistance, Sustained RNase H Activity, and Inhibition of KRAS Gene Expression.

The linear synthesis of 4'-C-aminoethoxy thymidine (AEoT) nucleoside phosphoramidite was accomplished using deoxythymidine as the starting material. This analog was incorporated into several oligonucleotides, the applicability of which as antisense oligonucleotides (ASOs) was then evaluated. The AEoT-modified DNA/RNA duplex exhibited improved thermal stability compared to unmodified and 4'-C-aminoethyl thymidine (4'-AET) modified heteroduplexes. The serum stability of AEoT-modified DNA was notably increased by several-folds compared to that of unmodified DNA. Furthermore, RNase H-dependent cleavage of the modified-DNA/RNA hybrids was found to be sustained. In addition, the modified antisense and unmodified oligonucleotides also displayed relatively comparable inhibition of the KRAS gene in human lung cancer cells. This study strengthens our understanding of the potential application of 4'-C-aminoethoxy-modified nucleotides as ASO therapeutics.

Synthesis of 4'-C-(aminoethyl)thymidine and 4'-C-[(N-methyl)aminoethyl]thymidine by a new synthetic route and evaluation of the properties of the DNAs containing the nucleoside analogs.

A gapmer-type antisense oligonucleotide is an oligonucleotide therapeutic that targets pathogenic mRNA directly, and it is expected to be a next-generation therapeutic drug. In this study, we designed and synthesized 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T) as a novel nucleoside analog and compared its properties with those of 4'-C-aminoethyl-thymidine (4'-AE-T). Furthermore, we designed a new synthetic route for 4'-C-aminoethyl-modified nucleosides and accomplished the synthesis of 4'-AE-T via a novel pathway with high total yield. DNA containing 4'-MAE-T analogs decreased RNA affinity slightly more than unmodified DNA and DNA containing 4'-AE-T, but significantly improved nuclease resistance compared to unmodified DNA in a solution containing bovine serum. In addition, the impact of 4'-MAE-T on DNA stability was higher than that of 4'-AE-T. Also, DNA containing these analogs can activate Escherichia coli-derived RNase H. Thus, 4'-MAE-T has the potential to be used in gapmer-type antisense nucleic acids as a suitable candidate for the development of therapeutic antisense oligonucleotides.

Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells.

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central "window" of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.