Oligonucleotide Sequences Research Articles

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Development of decoy oligonucleotide-warheaded chimeric molecules targeting STAT3

Proteolysis-targeting chimera (PROTAC) technology is a disruptive innovation in the drug development community, and over 20 PROTAC molecules are currently under clinical evaluation. These PROTAC molecules contain small-molecule warheads that bind to target proteins. Recently, oligonucleotide-warheaded PROTACs have emerged as a promising new tool to degrade DNA-binding proteins such as transcription factors. In this study, we applied an oligonucleotide-warheaded PROTAC technology to induce the degradation of signal transducer and activator of transcription 3 (STAT3), which is a hard-to-target protein. A double-stranded decoy oligonucleotide specific to STAT3 was conjugated to E3 binders (pomalidomide, VH032, and LCL161) to generate PROTAC molecules that recruited different E3 ubiquitin ligases cereblon (CRBN), von Hippel-Lindau (VHL), and inhibitor of apoptosis protein (IAP), respectively. One of the resulting PROTAC molecules, POM-STAT3, which recruits CRBN, potently induces STAT3 degradation. STAT3 degradation by POM-STAT3 was abolished by scrambling the oligonucleotide sequences of POM-STAT3 and by adding a double-stranded decoy oligonucleotide against STAT3 in a competitive manner, suggesting the significance of oligonucleotide sequences in STAT3 degradation. Moreover, POM-STAT3-induced STAT3 degradation was suppressed by the CRBN binder thalidomide, proteasome inhibitor bortezomib, E1 inhibitor MLN7243, and siRNA-mediated depletion of CRBN, indicating that STAT3 degradation is mediated by the ubiquitin-proteasome system, which involves CRBN as the responsible E3 ubiquitin ligase. Consistent with STAT3 degradation, NCI-H2087 cell viability was severely reduced following POM-STAT3 treatment. Thus, POM-STAT3 is a STAT3 degrader that potentially has cytocidal activity against cancer cells that are highly dependent on STAT3 signaling, which implies that inducing protein degradation by decoy oligonucleotide-warheaded PROTAC molecules could be harnessed to be therapeutic against oncogenic transcription factors.

Cell-based SELEX aptamer selection for electrochemical detection of Fluoribacter bozemanae bacteria

Fluoriobacter bozemanae (F. bozemanae) can cause Legionnaires' disease. The conventional methods for the detection of F. bozemanae are laborious, costly and has to be performed in centralized labs by skilled personnel. Therefore, there is an urgent need for rapid detection methods for F. bozemanae. The present work reports the first electrochemical aptasensor for the rapid and specific detection of the most virulent type of Legionellosis. Firstly, high-affinity aptamer was selected against the F. bozemanae using cell-based systematic evolution of ligands by exponential enrichment (Cell SELEX). The aptamers selections were based on 10 rounds of cell-SELEX with live F. bozemanae cells, and the counter-selection was performed against five other closely related Legionella sub-species. The selected oligonucleotide sequences were characterized using the dissociation constant (Kd). Apt-FB6 sequence gave a Kd value as low as 9.516 nM. This selected aptamer was integrated with electrochemical impedance spectroscopy (EIS). The impedimetric aptasensor for F. bozemanae exhibited a detection limit down to 10 CFU/mL. Finally, the aptasensor was tested against Legionella anisa, Legionella micdadei, Fluoriobacter dumoffii, and Legionella pneumophila serogroup 1 and 3. The biosensor didn't show significant cross-reactivity with the closely-related sub-species of Legionella. The selected aptasensor can be further exploited for further biosensing applications for clinical, environmental, and diagnostic purposes.

Monolithic affinity columns in 3D printed microfluidics for chikungunya RNA detection.

Mosquito-borne pathogens plague much of the world, yet rapid and simple diagnosis is not available for many affected patients. Using a custom stereolithography 3D printer, we created microfluidic devices with affinity monoliths that could retain, noncovalently attach a fluorescent tag, and detect oligonucleotide and viral RNA. We optimized the fluorescent binding and sample load times using an oligonucleotide sequence from chikungunya virus (CHIKV). We also tested the specificity of CHIKV capture relative to genetically similar Sindbis virus. Moreover, viral RNA from both viruses was flowed through capture columns to study the efficiency and specificity of the column for viral CHIKV. We detected ~107 loaded viral genome copies, which was similar to levels in clinical samples during acute infection. These results show considerable promise for development of this platform into a rapid mosquito-borne viral pathogen detection system.

Novel oligonucleotide-based sorbent for the selective extraction of cadmium from serum samples

The objective was to develop a sorbent functionalized with aptamers for the selective extraction of cadmium from biological samples. Two oligonucleotide sequences reported in literature as specific to cadmium were covalently grafted on activated Sepharose, with grafting yields of 45%. Once the supports packed in cartridges, a thorough study of the percolation conditions favoring Cd(II) retention was performed, demonstrating the importance of the nature of this medium. A high selectivity was reached when applying the optimal conditions as a recovery of 85% was obtained using the sorbent functionalized with one of the specific aptamers and only 1% on the control sorbent grafted with a scramble sequence. A high specificity was also obtained as recoveries for most of other ions were lower than 15%. The capacity of this oligosorbent estimated to 180 ng of Cd(II) for 30 mg of support was perfectly adapted to the trace analysis of Cd(II). The extraction procedure was then applied to a serum sample which was first subjected to acid precipitation. The initial concentration of cadmium in the serum was estimated to 1.83 µg/L using standard addition method and an extraction yield of 75 ± 1.6% was measured. Comparison of these results with those obtained without oligoextraction (recovery of 57%) showed a significant reduction of matrix effects in ICP-MS thanks to the use of the oligosorbent, underlining its interest for a more reliable quantification of Cd(II). This result was confirmed by performing the oligoextraction protocol on a certified serum.

Scaling logical density of DNA storage with enzymatically-ligated composite motifs

DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this work, we propose composite motifs, a framework that uses a mixture of prefabricated motifs as building blocks to reduce synthesis cost by scaling logical density. To write data, we introduce Bridge Oligonucleotide Assembly, an enzymatic ligation technique for synthesizing oligos based on composite motifs. To sequence data, we introduce Direct Oligonucleotide Sequencing, a nanopore-based technique to sequence short oligos, eliminating common preparatory steps like DNA assembly, amplification and end-prep. To decode data, we introduce Motif-Search, a novel consensus caller that provides accurate reconstruction despite synthesis and sequencing errors. Using the proposed methods, we present an end-to-end experiment where we store the text “HelloWorld” at a logical density of 84 bits/cycle (14–42× improvement over state-of-the-art).

Disposable and instrument-free nucleic acid lateral flow cassette for rapid and on-site identification of adulterated goat milk

Species identification has become a significant concern due to the growing use of food alternatives that may cause allergies and reduce nutritional value. To address the issue of fraudulent adulteration of goat milk products with cow milk, we have developed an affordable, portable, and user-friendly platform called microfluidic-integrated nucleic acid lateral flow strips (LFS). This platform enables simultaneous detection of components derived from both goats and cows in goat milk. In this study, we have introduced an innovative nucleic acid labeling method. The loop primers of loop-mediated isothermal amplification (LAMP) have been modified with amplification terminator spacer C3 and an oligonucleotide sequence, thus eliminating the requirement for costly antibodies in traditional nucleic acid LFS. This modification not only lowers costs but also enables multiple detections. Additionally, we have integrated the LAMP and LFS assay steps into a microfluidic chip, allowing convenient on-site detection while effectively preventing aerosol contamination of LAMP products. The testing process includes rapid DNA extraction, followed by a short nucleic acid addition and incubation for visualized results in about 50 min. This platform is user-friendly, requiring no specialized equipment or extensive training, making it suitable for rapid on-site detection of dairy products by personnel in diverse fields.

Sequence specificity defines the effectiveness of PPMOs targeting Pseudomonas aeruginosa.

Development of new therapeutics against antibiotic resistant pathogenic bacteria is recognized as a priority across the globe. We have reported using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) as species-specific antibiotics. The oligo sequences, 11 bases are designed to be complementary to specific essential genes near the Shine-Dalgarno site and inhibit translation. Here, we analyzed target specificity and the impact of genetic mutations on lead PPMOs targeting the rpsJ or acpP gene of Pseudomonas aeruginosa. Mutants in P. aeruginosa PAO1 were generated with four, two, or one base-pair mutations within the 11-base target sequence of the rpsJ gene. All mutants exhibited increased MICs compared to wild-type PAO1 when treated with the RpsJ PPMO, and the increase in the MICs was proportional to the number of base-pair mutations. Among single base-pair mutants, mutations in the middle of the sequence were more impactful than mutations in 5' or 3' end of the sequence. The increased MICs shown by the rpsJ mutants could be reversed by PPMOs designed to target the mutated rpsJ sequence. BALB/c mice infected intratracheally with mutants demonstrated increased lung burden when treated with RpsJ PPMO compared to wild-type PAO1-infected mice treated with RpsJ PPMO. Treating mice with a PPMOs designed to specifically target the mutant sequence was more effective against these mutant strains. These experiments confirm target specificity of two lead P. aeruginosa PPMOs and illustrate one potential mechanism of resistance that could emerge from an antisense approach.

Multicolor Hg2+ sensor based on the regulation of peroxidase activity of Fe/Co-MIL-88(NH2) and etching of gold nanobipyramids

The entry of Hg2+ into the human body can lead to serious damage to some organs and it is vital to construct some simple but sensitive Hg2+ assay methods. A multicolor sensor for Hg2+ was constructed depended on the inhibition of peroxidase activity of Fe/Co-MIL-88(NH2) mediated by DNA and color change caused by the etching of gold nanobipyramids (Au NBPs). The enzymatic activity of Fe/Co-MIL-88(NH2) can be inhibited by adsorbing the T-rich oligonucleotide sequence on its surface. Hg2+ can combine with thymine of the T-rich oligonucleotide sequence to constitute T-Hg2+-T complex, and cause the transforming of T-rich oligonucleotide sequence into three-dimensional structure and desorbed from Fe/Co-MIL-88(NH2) surface. Which results in the restoration of the peroxidase activity of Fe/Co-MIL-88(NH2), and then it catalyzes the 3,3′,5,5′-tetramethylbenzidine (TMB) to engender TMB2+, TMB2+ can etch Au NBPs, this accompanied with a peak shift of the UV–vis spectrum and recognizable color changes of the system. Semi-quantitative can be realized using the multicolor changes of the Au NBPs as the reading mode. Furthermore, quantitative sensing can be achieved by measuring the UV–vis peak shift of the system. The linear response range is 1.0 nM–10 µM, and the detection limit (LOD) is 0.28 nM.

In Vivo Imaging of [60]Fullerene-Based Molecular Spherical Nucleic Acids by Positron Emission Tomography.

18F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factor receptor 2 (HER2) mRNA antisense oligonucleotide sequence with a native phosphodiester and phosphorothioate backbone, were synthesized, site-specifically labeled with a positron emitting fluorine-18 and intravenously administrated via tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistribution of the MSNAs was monitored in vivo by positron emission tomography/computed tomography (PET/CT) imaging. MSNA with a native phosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediated degradation, whereas the corresponding phosphorothioate analogue (MSNA-PS) with improved enzymatic stability showed an interesting biodistribution profile in vivo. One hour after the injection, majority of the radioactivity was observed in spleen and liver but also in blood with an average tumor-to-muscle ratio of 2. The prolonged radioactivity in blood circulation may open possibilities to the targeted delivery of the MSNAs.

Flexible genosensors based on polypyrrole and graphene quantum dots for PML/RARα fusion gene detection: A study of acute promyelocytic leukemia in children

Acute promyelocytic leukemia (APL) in children is associated with a favorable initial prognosis. However, minimal residual disease (MRD) follow-up remains poorly defined, and relapse cases are concerning due to their recurrent nature. Thus, we report two electrochemical flexible genosensors based on polypyrrole (PPy) and graphene quantum dots (GQDs) for label-free PML-RARα oncogene detection. Atomic force microscopy (AFM), scanning electron microscope (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to characterize the technological biosensor development. M7 and APLB oligonucleotide sequences were used as bioreceptors to detect oncogenic segments on chromosomes 15 and 17, respectively. AFM characterization revealed heterogeneous topographical surfaces with maximum height peaks for sensor layers when tested with positive patient samples. APLB/Genosensor exhibited a percentage change in anode peak current (ΔI) of 423 %. M7/Genosensor exhibited a ΔI of 61.44 % for more concentrated cDNA samples. The described behavior is associated with the biospecific recognition of the proposed biosensors. Limits of detection (LOD) of 0.214 pM and 0.677 pM were obtained for APLB/Genosensor and M7/Genosensor, respectively. The limits of quantification (LOQ) of 0.648 pM and 2.05 pM were estimated for APLB/Genosensor and M7/Genosensor, respectively. The genosensors showed reproducibility with a relative standard deviation of 7.12 % for APLB and 1.18 % for M7 and high repeatability (9.89 % for APLB and 1.51 % for M7). In addition, genetic tools could identify the PML-RARα oncogene in purified samples, plasmids, and clinical specimens from pediatric patients diagnosed with APL with high bioanalytical performance. Therefore, biosensors represent a valuable alternative for the clinical diagnosis of APL and monitoring of MRD with an impact on public health.

Spatially-Encoding Hydrogels with DNA to Control Cell Signaling.

Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially-encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, we introduce a method to pattern multiple oligonucleotide sequences in hydrogels using thiol-yne photochemistry. Rapid hydrogel photo-patterning of hydrogels with micron resolution DNA features (∼1.5µm) and control over DNA density are achieved over centimeter-scale areas using mask-free digital photolithography. Sequence-specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Finally, localized cell signaling is shown using patterned protein-DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron-resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially encoded cellular signaling environments. This article is protected by copyright. All rights reserved.

Integration of in-cassette lysis, purification, and lateral flow strips-based sensor for rapid and on-site detection of yak milk adulteration

Amplification-based nucleic acid testing has become a widely used method for food authenticity analysis. However, due to the need for specialized instrumentation, technical expertize, and trained operators, routine nucleic acid analysis can only be performed in a laboratory setting. For perishable products like yak milk, rapid and accurate detection of dairy components on-site is crucial for the automated management of the production process and improving the milk's quality at dairies. As a feasibility study, we developed an assay strategy for the simultaneous qualitative detection of yak and cow derived components in yak milk. We presented a handheld integrated cassette that combined rapid extraction and purification of dairy nucleic acids, loop-mediated isothermal amplification (LAMP), and visual detection of lateral flow strips (LFS). To enable visual detection of LAMP amplification products on LFS, we utilized a unique primer modification method that labeled the amplification terminator and single oligonucleotide sequence at the 5′ end of the loop primer. This modification avoids the use of antibodies and conventional fluorescein/biotin. The cassette allowed for DNA transfer, fluid drive, and liquid mixing by magnetic force and gravity alone, without the need for complex pumps and valves. The disposable cassette is easy to use, costs less than $1 to produce, has low instrument dependency, and is well suited to on-site detection. The platform can detect 0.1% of cow milk adulteration in yak milk within 50 min. This fully integrated "sample in-result out" detection procedure can also be extended to other detection areas.

Triplex-colored nucleic acid lateral flow strip and multiplex polymerase chain reaction coupled method for quantitative identification of beef, pork and chicken

Meat adulteration is one of the worldwide challenges for food quality control. Now, we developed a multiplex PCR and nucleic acid lateral flow strip (mPCR-NALFS) integrated method that employs triplex-color latex microspheres (LMs) as visual labels for simultaneous identification of beef, pork and chicken DNA. First, three primer pairs for beef, pork and chicken DNA are tailed with specific oligonucleotide sequences (anti-recognition/anti-detection/anti-control probes) as swing tags. Then, double-stranded amplicons obtained after mPCR amplification were flanked by tags. Finally, these amplicons can accurately hybridize with the pre-immobilized recognition probes on the conjugate pad, detection probes on the test lines and control probes on the control line, respectively. The LMs and recognition probe modified LMs were characterized by scanning electron microscopy, ultraviolet-visible spectroscopy and Fourier Transform Infrared spectroscopy. This strip can quantitatively detect beef, pork and chicken DNA within 10 min without compromising on accuracy compared to the electrophoresis. Specificity analysis revealed no cross-reactivity with meat derived from beef, pork, chicken, duck, mutton, rabbit, goose, ostrich, camel and horse meat. As low as 0.1 % (wt%) of beef, pork and chicken in the simulated adulterated meats were co-detected. This assay was successfully employed for adulteration identification in 28 commercial beef and lamb products.

Molecular cytogenetic maps provide new insights into the differences between hybrid Citrus microcarpa and lemons

Citrus microcarpa is one of the most popular species for flavoring and industrialized juice production, making it an increasingly important economic crop. At present, C. microcarpa has been mistakenly regarded as a type of lemon in China. The lack of a simple identification method for hybrid C. microcarpa is the major obstacle limiting its comprehensive recognition and utilization. In this study, firstly nine pairs of chromosomes were marked by eight oligo pools (OPs) in C. microcarpa, C. aurantiifolia, and C. limon, which showed that oligonucleotide (oligo) sequences are highly conserved within the genomes of the citrus family, suggesting that eight OPs and nine chromosome painting probes (CPPs) can be used as universal probes for the identification of each chromosome in citrus plants. Then we developed a method for the identification of C. microcarpa by using fluorescence in situ hybridization (FISH) mapping and Chromoycin A3 (CMA) staining. Moreover, we constructed the molecular cytogenetic maps containing the physical positions of eight OPs, 45S rDNA, 5S rDNA, CL, CMA banding and fragile sites in C. microcarpa, C. aurantiifolia, and C. limon, which visualize the significant differences among the three Citrus species. Combining the highly different morphological and cytological characteristics of C. microcarpa with C. aurantiifolia and C. limon, it is clear that C. microcarpa is a Citrus hybrid species, not a lemon species. Overall, this study provides a better understanding of C. microcarpa from the molecular cytogenetic perspective.

BIC Codes: Bit Insertion-Based Constrained Codes With Error Correction for DNA Storage

In this paper, we propose a new coding algorithm for DNA storage over both error-free and error channels. For the error-free case, we propose a constrained code called bit insertion-based constrained (BIC) code. BIC codes convert a binary data sequence to multiple oligo sequences satisfying the maximum homopolymer run (i.e., run-length (RL)) constraint by inserting dummy bits. We show that the BIC codes nearly achieves the capacity in terms of information density while the simple structure of the BIC codes allows linear-time encoding and fast parallel decoding. Also, by combining a balancing technique with the BIC codes, we obtain the constrained coding algorithm to satisfy the GC-content constraint as well as the RL constraint. Next, for DNA storage channel with errors, we integrate the proposed constrained coding algorithm with a rate-compatible low-density parity-check (LDPC) code to correct errors and erasures. Specifically, we incorporate LDPC codes adopted in the 5G new radio standard because they have powerful error-correction capability and appealing features for the integration. Simulation results show that the proposed integrated coding algorithm outperforms existing coding algorithms in terms of information density and error correctability.

Highly sensitive detection of microRNA-21 by nitrogen-doped carbon dots-based ratio fluorescent probe via nuclease-assisted rolling circle amplification strategy

Highly sensitive and selective detection of microRNA-21 (miRNA-21) in biological samples is critical for the disease diagnosis and cancer treatment. In this study, a nitrogen-doped carbon dots (N-CDs)-based ratio fluorescence sensing strategy was constructed for miRNA-21 detection with high sensitivity and excellent specificity. Bright-blue N-CDs (λex/λem = 378 nm/460 nm) were synthesized by facile one-step microwave-assisted pyrolysis method by using uric acid as the single precursor, and the absolute fluorescence quantum yield and fluorescence lifetime of N-CDs were 35.8% and 5.54 ns separately. The padlock probe hybridized with miRNA-21 firstly and then was cyclized by T4 RNA ligase 2 to form a circular template. At the present of dNTPs and phi29 DNA polymerase, the oligonucleotide sequence in miRNA-21 was prolonged to hybridize with the surplus oligonucleotide sequences in circular template, generating long and reduplicated oligonucleotide sequences containing abundant guanine nucleotides. Separate G-quadruplex sequences were generated after the addition of Nt.BbvCI nicking endonuclease, and then hemin bound with G-quadruplex sequence to construct the G-quadruplex DNAzyme. Such G-quadruplex DNAzyme catalyzed the redox reaction of o-phenylenediamine (OPD) with H2O2, finally producing the yellowish-brown 2,3-diaminophenazine (DAP) (λem = 562 nm). Due to the inner filter effect between N-CDs and DAP, the ratio fluorescence signal of DAP with N-CDs was utilized for sensitive detection of miRNA-21 with detection limit of 0.87 pM. Such approach has practical feasibility and excellent specificity for miRNA-21 analysis during highly homological miRNA family in HeLa cell lysates and human serum samples.

Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100–10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.

Generation of DNA Aptamers with Functional Activity in Mammalian Cells by Mimicking Retroviruses.

DNA aptamers are single-stranded DNA oligonucleotide sequences that bind to specific targets with high affinity. Currently, DNA aptamers can be produced only by in vitro synthesis. It is difficult for DNA aptamers to have a sustained impact on intracellular protein activity, which limits their clinical application. In this study, we developed a DNA aptamer expression system to generate DNA aptamers with functional activity in mammalian cells by mimicking retroviruses. Using this system, DNA aptamers targeting intracellular Ras (Ra1) and membrane-bound CD71 (XQ2) were successfully generated in cells. In particular, the expressed Ra1 not only specifically bound to the intracellular Ras protein but also inhibited the phosphorylation of downstream ERK1/2 and AKT. Furthermore, by inserting the DNA aptamer expression system for Ra1 into a lentivirus vector, the system can be delivered into cells and stably produce Ra1 over time, resulting in the inhibition of lung cancer cell proliferation. Therefore, our study provides a novel strategy for the intracellular generation of DNA aptamers with functional activity and opens a new avenue for the clinical application of intracellular DNA aptamers in disease treatment.

An enzyme cascade enables production of therapeutic oligonucleotides in a single operation.

Therapeutic oligonucleotides have emerged as a powerful drug modality with the potential to treat a wide range of diseases; however, the rising number of therapies poses a manufacturing challenge. Existing synthetic methods use stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. We report a biocatalytic approach to efficiently produce oligonucleotides in a single operation where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks and aqueous conditions. We demonstrate the versatility of this methodology through the synthesis of clinically relevant oligonucleotide sequences containing diverse modifications.