Short Single-stranded Oligonucleotides Research Articles

Identification and bioinformatic characterization of a serum miRNA signature for early detection of laryngeal squamous cell carcinoma

BackgroundThe growing understanding of cancer biology and the establishment of new treatment modalities has not yielded the expected results in terms of survival for Laryngeal Squamous Cell Cancer (LSCC). Early diagnosis, as well as prompt identification of patients with high risk of relapse would ensure greater chance of therapeutic success. However, this goal remains a challenge due to the absence of specific biomarkers for this neoplasm.MethodsSerum samples from 45 LSCC patients and 23 healthy donors were collected for miRNA expression profiling by TaqMan Array analysis. Additional 20 patients and 42 healthy volunteers were included for the validation set, reaching an equal number of clinical samples for each group. The potential diagnostic ability of the such identified three-miRNA signature was confirmed by ROC analysis. Moreover, each miRNA was analyzed for the possible correlation with HNSCC patients’ survival and TNM status by online databases Kaplan–Meier (KM) plotter and OncomiR. In silico analysis of common candidate targets and their network relevance to predict shared biological functions was finally performed by PANTHER and GeneMANIA software.ResultsWe characterized serum miRNA profile of LSCC patients identifying a novel molecular signature, including miR-223, miR-93 and miR-532, as circulating marker endowed with high selectivity and specificity. The oncogenic effect and the prognostic significance of each miRNA was investigated by bioinformatic analysis, denoting significant correlation with OS. To analyse the molecular basis underlying the pro-tumorigenic role of the signature, we focused on the simultaneously regulated gene targets—IL6ST, GTDC1, MAP1B, CPEB3, PRKACB, NFIB, PURB, ATP2B1, ZNF148, PSD3, TBC1D15, PURA, KLF12—found by prediction tools and deepened for their functional role by pathway enrichment analysis. The results showed the involvement of 7 different biological processes, among which inflammation, proliferation, migration, apoptosis and angiogenesis.ConclusionsIn conclusion, we have identified a possible miRNA signature for early LSCC diagnosis and we assumed that miR-93, miR-223 and miR-532 could orchestrate the regulation of multiple cancer-related processes. These findings encourage the possibility to deepen the molecular mechanisms underlying their oncogenic role, for the desirable development of novel therapeutic opportunities based on the use of short single-stranded oligonucleotides acting as non-coding RNA antagonists in cancer.

Medicinal Chemistry of Antisense Oligonucleotides for Therapeutic Use in SARS-CoV-2: Design Strategies and Challenges for Targeted Delivery

Background: The evolution of novel Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2) strains with greater degrees of infectivity, resistance to vaccine-induced acquired immunity, and more severe morbidity have contributed to the recent spread of COVID-19. In light of this, novel therapeutic alternatives with improved effectiveness and fewer side effects have become a necessity. Despite many new or repurposed antiviral agents recommended for Coronavirus disease (COVID-19) therapy, this objective remains unfulfilled. Under these circumstances, the scientific community holds the significant responsibility to develop classes of novel therapeutic modalities to combat SARS-CoV-2 with the least harmful side effects. Objective: Antisense Oligonucleotides (ASOs) are short single-stranded oligonucleotides that allow the specific targeting of RNA, leading to its degradation. They may also prevent cellular factors or machinery from binding to the target RNA. It is possible to improve the pharmacokinetics and pharmacodynamics of ASOs by chemical modification or bioconjugation, which may provide conditions for customization of a particular clinical target. This study aimed to outline the potential use of ASOs in the treatment of COVID-19 disease, along with the use of antisense stabilization and transfer methods, as well as future challenges and limitations. Methods: We have reviewed the structure and properties of ASOs containing nucleobase, sugar, or backbone modifications, and provided an overview of the therapeutic potential, delivery challenges, and strategies of ASOs in the treatment of COVID-19. Results: The first-line therapy for COVID-19-infected individuals, as well as the development of oligonucleotide-based drugs, warrants further investigation. Chemical changes in the oligonucleotide structure can affect the biological processes. These chemical alterations may lead to enhanced potency, while changing the pharmacokinetics and pharmacodynamics. Conclusion: ASOs can be designed to target both coding and non-coding regions of the viral genome to disrupt or completely degrade the genomic RNA and thereby eliminate SARS-CoV-2. They may be very effective in areas, where vaccine distribution is challenging, and they may be helpful for future coronavirus pandemics.

Abstract LB432: Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme

Abstract Glioblastoma Multiforme (GBM) is one of the most aggressive and lethal brain tumor types with a five-year survival rate of 6.9%. In 2023, more than 14,490 Americans were expected to receive a diagnosis. Although great progress has been achieved in protein target identification and treatment over the last few years, current treatment options are limited, and tumor recurrence occurs at a high rate due to treatment resistance. Aptamers, short single-stranded oligonucleotide ligands with high-affinity binding to molecular targets, are well suited tools for exploring novel targets in highly heterogeneous tumors with complex etiologies like GBM, where clinically relevant biomarkers are limited. In this study, aptamer-based cancer-specific drug target identification was performed by Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) Biotargeting System. In short, a GBM-specific library was enriched through a Systematic Evolution of Ligands by EXponential enrichment (SELEX) process using micro dissected Formalin-Fixed Paraffin-Embedded (FFPE) tissues from GBM patients, along with whole body normal tissue for counterselection. Microdissection of tissue allows the partitioning of different types of cells to focus on specific cells of interest. The enriched libraries have been evaluated on clinical GBM and normal brain control samples through aptamer mediated tissue staining, resulting in preferential binding of enriched libraries compared to controls in the vast majority of GBM patients with little binding to normal brain samples. The final library was subsequently applied in an in-house developed library-based affinity pull-down assay using lysates from FFPE tissue of the same group of GBM patients and normal brain samples, followed by unbiased Mass Spectrometry for protein target identification. Combining the results from this proteomics analysis and data from the internal Whole Transcriptome Sequencing (WTS) database, we were able to identify several targets that are highly expressed in GBM and other cancer lineages. Antibody staining provided further validation of the expression levels and the cellular localization. The targets identified in this study include not only potential novel therapeutic targets, but also well-studied biomarkers and targets with available drugs already in clinical trials, demonstrating the platform has great potential for identifying high affinity binding and selectivity compared to traditional proteomics and other methods in drug target discovery. Citation Format: Xixi Wei, Myunggi An, Radhika Santhanam, Barbara Richardson, Lucas Vu, Justin Saul, Anna Walton, Michael Gee, Daniel Martin, Amanda Hubbard, Alexis Rodin, Matthew Rosenow, Tassilo Hornung. Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB432.

Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications.

Systematic evolution of ligands by exponential enrichment (SELEX) has been used to discover thousands of aptamers since its development in 1990. Aptamers are short single-stranded oligonucleotides capable of binding to targets with high specificity and selectivity through structural recognition. While aptamers offer advantages over other molecular recognition elements such as their ease of production, smaller size, extended shelf-life, and lower immunogenicity, they have yet to show significant success in real-world applications. By analyzing the importance of structured library designs, reviewing different SELEX methodologies, and the effects of chemical modifications, we provide a comprehensive overview on the production of aptamers for applications in drug delivery systems, therapeutics, diagnostics, and molecular imaging.

Aptamer-Based Recognition of Breast Tumor Cells: A New Era for Breast Cancer Diagnosis.

Breast cancer is one of the leading causes of death among women worldwide and can be classified into four major distinct molecular subtypes based on the expression of specific receptors. Despite significant advances, the lack of biomarkers for detailed diagnosis and prognosis remains a major challenge in the field of oncology. This study aimed to identify short single-stranded oligonucleotides known as aptamers to improve breast cancer diagnosis. The Cell-SELEX technique was used to select aptamers specific to the MDA-MB-231 tumor cell line. After selection, five aptamers demonstrated specific recognition for tumor breast cell lines and no binding to non-tumor breast cells. Validation of aptamer specificity revealed recognition of primary and metastatic tumors of all subtypes. In particular, AptaB4 and AptaB5 showed greater recognition of primary tumors and metastatic tissue, respectively. Finally, a computational biology approach was used to identify potential aptamer targets, which indicated that CSKP could interact with AptaB4. These results suggest that aptamers are promising in breast cancer diagnosis and treatment due to their specificity and selectivity.

APTAMERS: NANOMATERIALS AS A POTENTIAL AGENT FOR ANTIVIRAL THERAPEUTIC DRUG DELIVERY DEVELOPMENT: A SYSTEMATIC LITERATURE REVIEW

Chemotherapeutic experts have been utilised to cure a variety of disorders, but their practical application is restricted due to their regrettable selectivity and outrageous fundamental optional effects. Short single-stranded DNA or RNA oligonucleotides known as aptamers are released from randomised libraries and have strong propensity and differentiation towards targets like antibodies as well as characterised structures and ties to targets like proteins. They commonly suppress protein interactions while restricting proteins, which may elicit positive effects like threat. Aptamers have recently demonstrated their amazing promise for use in medicines, biosensors, and bioimaging thanks to a number of advantages, such as minimal immunogenicity, simplicity of giant degree blend, low pack to-bunch collection, genuinely substance modification, and programmability. At any rate, the steady for the most part accomplishment speed of aptamer is far from being brilliant, despite everything needs to overwhelm the gigantic obstruction in propensity, constancy for utilitarian application, explicit illness cell affirmation. The sensible method of controlling the binding execution of aptamers and dealing with their show in the practical application is of great significance and these single-abandoned DNA or RNA aptamers could outline with astoundingly poisonous chemotherapy drugs, hurts, strong RNAs or different particles as novel aptamer-drug structures, which are prepared to do endlessly out working on the obliging plentifulness and decreasing the critical danger of solutions and have unprecedented possible in living spaces for appointed ailment treatment. In this survey, we have extensively covered and summarised the ongoing improvements in the aptamer-drug structure philosophy for designated drug transport in the assessment methodologies of aptamers for unambiguous disease biomarkers. A modified strategy utilising aptamers could be a reliable system for quick and precise advancement of biopharmaceutics for use in infection-related treatment, especially in light of the enormous advances in modernised thinking for protein and RNA structure conjectures. Additionally, the likelihood of future conception is also summarised.

Aptamers as Potential Therapeutic Tools for Ovarian Cancer: Advancements and Challenges

Ovarian cancer (OC) is the most common lethal gynecologic cause of death in women worldwide, with a high mortality rate and increasing incidence. Despite advancements in the treatment, most OC patients still die from their disease due to late-stage diagnosis, the lack of effective diagnostic methods, and relapses. Aptamers, synthetic, short single-stranded oligonucleotides, have emerged as promising anticancer therapeutics. Their ability to selectively bind to target molecules, including cancer-related proteins and receptors, has revolutionized drug discovery and biomarker identification. Aptamers offer unique insights into the molecular pathways involved in cancer development and progression. Moreover, they show immense potential as drug delivery systems, enabling targeted delivery of therapeutic agents to cancer cells while minimizing off-target effects and reducing systemic toxicity. In the context of OC, the integration of aptamers with non-coding RNAs (ncRNAs) presents an opportunity for precise and efficient gene targeting. Additionally, the conjugation of aptamers with nanoparticles allows for accurate and targeted delivery of ncRNAs to specific cells, tissues, or organs. In this review, we will summarize the potential use and challenges associated with the use of aptamers alone or aptamer–ncRNA conjugates, nanoparticles, and multivalent aptamer-based therapeutics for the treatment of OC.

Telomerase RNA-based aptamers restore defective myelopoiesis in congenital neutropenic syndromes

Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus facilitating myeloid gene expression. The CR4/CR5 domain of TERC is known to play this role, since a mutation of this domain found in dyskeratosis congenita (DC) patients decreases its affinity for RNA Pol II, impairing its myelopoietic activity as a result. In this study, we report that two aptamers, short single-stranded oligonucleotides, based on the CR4/CR5 domain were able to increase myelopoiesis without affecting erythropoiesis in zebrafish. Mechanistically, the aptamers functioned as full terc; that is, they increased the expression of master myeloid genes, independently of endogenous terc, by interacting with RNA Pol II and with the terc-binding sequences of the regulatory regions of such genes, enforcing their transcription. Importantly, aptamers harboring the CR4/CR5 mutation that was found in DC patients failed to perform all these functions. The therapeutic potential of the aptamers for treating neutropenia was demonstrated in several preclinical models. The findings of this study have identified two potential therapeutic agents for DC and other neutropenic patients.

Universal Method Based on Layer-by-Layer Assembly for Aptamer-Based Sensors for Small-Molecule Detection.

Development of a fast and accurate pesticide analysis system is a challenging task, as a large amount of commonly used pesticide has negative effects on humans' health. Detection of pesticide residues is crucial for food safety management and environmental protection. Aptamers─short single-stranded oligonucleotides (RNA or DNA) selected by aptamer selection method SELEX─can selectively bind to their target pesticide molecules with high affinity. Thus, in the present study, we developed an electrochemical biosensor based on aptamers to detect the commonly used pesticide, glyphosate. Carbon fibers were used as the platform to assemble polyelectrolyte layers with the incorporated aptamers selectively binding with glyphosate molecules for electrochemical detection. The best limit of detection of 0.3 μM was achieved at open-circuit potential measurements, which is comparable to the current need in detection of glyphosate. The developed method can be implemented into existing systems for the determination of pesticides on farms to control residual concentrations of glyphosate in soil and water.

Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo

Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo

Polycation Architecture Affects Complexation and Delivery of Short Antisense Oligonucleotides: Micelleplexes Outperform Polyplexes.

Herein, we examine the complexation and biological delivery of a short single-stranded antisense oligonucleotide (ASO) payload with four polymer derivatives that form two architectural variants (polyplexes and micelleplexes): a homopolymer poly(2-dimethylaminoethyl methacrylate) (D), a diblock polymer poly(ethylene glycol)methylether methacrylate-block-poly(2-dimethylaminoethyl methacrylate) (ObD), and two micelle-forming variants, poly(2-dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (DB) and poly(ethylene glycol)methylether methacrylate-block-poly(2-dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (ObDB). Both polyplexes and micelleplexes complexed ASOs, and the incorporation of an Ob brush enhances colloidal stability. Micellplexes are templated by the size and shape of the unloaded micelle and that micelle-ASO complexation is not sensitive to formulation/mixing order, allowing ease, versatility, and reproducibility in packaging short oligonucleotides. The DB micelleplexes promoted the largest gene silencing, internalization, and tolerable toxicity while the ObDB micelleplexes displayed enhanced colloidal stability and highly efficient payload trafficking despite having lower cellular uptake. Overall, this work demonstrates that cationic micelles are superior delivery vehicles for ASOs denoting the importance of vehicle architecture in biological performance.

Development of DNA aptamers specific for small therapeutic peptides using a modified SELEX method.

Aptamers are short single-stranded DNA or RNA oligonucleotides capable of binding with high affinity and specificity to target molecules. Because of their durability and ease of synthesis, aptamers are used in a wide range of biomedical fields, including the diagnosis of diseases and targeted delivery of therapeutic agents. The aptamers were selected using a process called systematic evolution of ligands by exponential enrichment (SELEX), which has been improved for various research purposes since its development in 1990. In this protocol, we describe a modified SELEX method that rapidly produces high aptamer screening yields using two types of magnetic beads. Using this method, we isolated an aptamer that specifically binds to an antimicrobial peptide. We suggest that by conjugating a small therapeutic-specific aptamer to a gold nanoparticle-based delivery system, which enhances the stability and intracellular delivery of peptides, aptamers selected by our method can be used for the development of therapeutic agents utilizing small therapeutic peptides.

Identification of G-quadruplex anti-Interleukin-2 aptamer with high specificity through SELEX stringency

Aptamers are short single-stranded oligonucleotides capable of binding to various targets with high specificity and affinity. This study aimed to identify an aptamer against mouse interleukin-2 (mIL-2) as one of the most important cytokines in autoimmune diseases for diagnostic and therapeutic purposes. For this purpose, 14 SELEX rounds were performed on recombinant mIL-2 with high stringency. The dot blot and flow cytometry techniques were conducted to determine affinity, dissociation constant (Kd), specificity, and SELEX rounds screening. The stringency of rounds was considered based on aptamer/target incubation time, washing steps, and target proteins. Finally, the aptamer's structure was mapped and predicted by M-fold and QGRS Mapper web-based software. After 14 rounds, the flow cytometry analysis revealed that the 11th round was a proper round. The high-affinity aptamers M20 and M15 were chosen for their ability to bind mIL-2. According to DNA folding software, M20 and M15 aptamers had G-quadruplex and stem-loop structures, respectively. The M20 aptamer affinity was greater than M15, and its predicted Kd was 91 nM. A simple SELEX protocol with round stringency was explained to identify DNA aptamers against protein targets. The reported G-quadruplex aptamer might have potential diagnostic or therapeutic application in IL-2–related disorders.

Lifetime of glass nanopores in a PDMS chip for single-molecule sensing

SummaryNanopore sensing is an emerging technology that has many biosensing applications ranging from DNA sequencing using biological pores to biomolecular analysis using solid-state pores. Solid-state nanopores that are more stable are an attractive choice for biosensing applications. Still, biomolecule interactions with the nanopore surface reduce nanopore stability and increase usage costs. In this study, we investigated the biosensing capability for 102 quartz glass nanopores with a diameter of 11–18 nm that were fabricated using laser-assisted capillary pulling. Nanopores were assembled into multiple microfluidic chips that were repeatedly used for up to 19 weeks. We find that using vacuum storage combined with minimal washing steps improved the number of use cycles for nanopores. The single-molecule biosensing capability over repeated use cycles was demonstrated by quantitative analysis of a DNA carrier designed for detection of short single-stranded DNA oligonucleotides.

The Aptamer Functionalized Nanocomposite Used for Prostate Cancer Diagnosis and Therapy

Prostate cancer is one of the major malignancies that threaten men’s health all over the world. Due to the lack of specific symptoms and signs in the early stage, as well as the limitations of existing detection methods, it is difficult to achieve early diagnosis for prostate cancer. As short single-stranded oligonucleotides (DNA or RNA) with specific 3D structure which can be produced using an in vitro selection process termed systematic evolution of ligands by exponential enrichment (SELEX), aptamers can specifically bind to the corresponding targets. They have become a class of novel targeting ligand for accurate diagnosis and effective treatment of cancer. Owing to distinctive physicochemical features, and some other special properties such as easy modifiability, good biocompatibility, being easily coupled with other ligands, nanomaterials are extensively used in biological medical field research. Enlighteningly, the combination of aptamers with nanomaterials, including metal nanoparticles, nanosilica, quantum dots, and carbon nanomaterials, can enhance the ability of nanomaterials to recognize tumor cells, which is beneficial to overcome the shortcomings such as low sensitivity in early detection and lack of specificity of traditional antineoplastic drugs, thus, clinically helpful to improve the early metaphase diagnosis rate, providing a technical guarantee for the “personalized treatment” strategy for prostate cancer. Herein, we mainly review the basic and applied research of aptamer functionalized nanocomposite in prostate cancer diagnosis and treatment, including biosensing, bioimaging, and cancer therapy, hoping to provide new ideas for prostate cancer diagnosis and treatment.

Optimized two-step electroporation process to achieve efficient nonviral-mediated gene insertion into primary T cells.

The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock‐in approaches using homology‐directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene‐edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double‐stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator‐like effector nuclease (TALEN)‐mediated gene editing in primary T cells to improve nonviral gene knock‐in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single‐stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time‐controlled two‐step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T‐cell receptor alpha constant region (TRAC) locus to generate ‘off‐the shelf’ CAR‐T cells.

Applications of Aptamers in the Diagnosis and Treatment of Ovarian Cancer: Progress From 2016 to 2020.

Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides selected from a random single-stranded nucleic acid library using systematic evolution of ligands by exponential enrichment technology. To allow them to bind to molecular targets with the same specificity and precision as that of antibodies, aptamers are folded into secondary or tertiary structures. However, compared to antibodies, aptamers are not immunogenic and are easier to synthesize. Furthermore, they are chemically modified, which protects them from degradation by nucleases. Hence, due to their stability and favorable targeting ability, aptamers are promising for the diagnosis and treatment of diseases. Ovarian cancer has the worst prognosis among all gynecological diseases and is usually diagnosed at the medium and advanced stages due to its nonspecific symptoms. Relapse is common, even if patients receive a standard therapeutic regimen including surgery and chemotherapy; simultaneously, drug resistance and adverse effects are reported in a several patients. Therefore, the safer and more efficient diagnostic and treatment method for ovarian cancer is imperative. Scientists have been trying to utilize aptamer technology for the early diagnosis and accurate treatment of ovarian cancer and some progress has been made in this field. This review discusses the screening of nucleic acid aptamers by targeting ovarian cancer cells and the application of aptamers in the diagnosis and treatment of ovarian cancer.

Selection of DNA Aptamers Recognizing EpCAM-Positive Prostate Cancer by Cell-SELEX for in vitro and in vivo MR Imaging

BackgroundThe sensitive and specific detection of pathogenic cells is important in tumor diagnosis at an early stage. Aptamers are short single-stranded oligonucleotides evolved from systematic evolution of ligands by exponential enrichment (SELEX). It has been proved that aptamers can interact with cognate target molecules with high affinity and specificity and have great potential in the development of medical imaging at molecular level.PurposeTo select epithelial cell adhesion molecule (EpCAM) specific aptamers targeting prostate cancer and further to conjugate aptamers with GoldMag nanoparticles (a typical iron oxide core/gold shell structure) to construct magnetic molecular probes for medical imaging.MethodsEpCAM-specific aptamers were selected by Cell-SELEX. The enrichment of specific aptamer candidates was monitored by flow cytometric analysis. Aptamers were further conjugated with GoldMag nanoparticles to construct magnetic molecular probes. The affinity and specificity of aptamer candidates and aptamer-conjugated GoldMag nanoparticles were evaluated. The MR imaging of aptamer-conjugated GoldMag nanoparticles to prostate cancer was further explored in vitro and in vivo.ResultsAfter 12 rounds of selection, aptamer candidates Eppc6 and Eppc14 could specifically target three types of prostate cancer cells, revealing a high affinity of Eppc6 and Eppc14. Moreover, aptamer-conjugated GoldMag nanoparticles not only exhibited good affinity to different prostate cancer cells but also produced strong T2WI signal intensity reduction distinguished from peritumoral tissue in MRI, indicating that the molecular probes possess both the affinity properties of EpCAM-specific aptamer and the superparamagnetic features of iron oxide.ConclusionOur study indicates that aptamer Eppc6 and Eppc14 can recognize prostate cancer cells and tissues. The aptamer-conjugated GoldMag nanoparticles constructed in the study can be used as a molecular imaging agent for detection of PCa in MRI.

An electrochemiluminescence resonance energy transfer biosensor for the detection of circulating tumor DNA from blood plasma

SummaryA liquid biopsy is a noninvasive approach for detecting double-stranded circulating tumor DNA (ctDNA) of 90–320 nucleotides in blood plasma from patients with cancer. Most techniques employed for ctDNA detection are time consuming and require expensive DNA purification kits. Electrochemiluminescence resonance energy transfer (ECL-RET) biosensors exhibit high sensitivity, a wide response range, and are promising for straightforward sensing applications. Until now, ECL-RET biosensors have been designed for sensing short single-stranded oligonucleotides of less than 45 nucleotides. In this work, an ECL-RET biosensor comprising graphitic carbon nitride quantum dots was assessed for the amplification-free detection in the blood plasma of DNA molecules coding for the EGFR L858R mutation, which is associated with non-small-cell lung cancer. Following a low-cost pre-treatment, the highly specific ECL-RET biosensor quantified double-stranded EGFR L858R DNA of 159 nucleotides diluted into the blood within a linear range of 0.01 fM to 1 pM, demonstrating its potential for noninvasive biopsies.

Construction of Aptamer-Based Molecular Beacons with Varied Blocked Structures and Targeted Detection of Thrombin.

A kind of blocked aptamer-functionalized molecular beacon (MB) was designed as fluorescence sensors to detect thrombins by binding-induced "turn on" structural transformation. Three MBs named MB(8 + 8), MB(15 + 8), and MB(15 + 6) consisted of two single-stranded oligonucleotides. One long single-stranded oligonucleotide (abbreviated as SS) contained a thrombin aptamer sequence and was modified with a fluorescence group and quenching group on each end side. Another short single-stranded oligonucleotide (written as cDNA) was partially complementary to the long SS. It was interesting to find that the complementary sequence length of cDNA greatly influenced the structure of the MBs. The construction of MB experiments proved that MB(8 + 8) and MB(15 + 8) could form the quenching MBs but MB(15 + 6) could not. MB(8 + 8) was composed of a SS strand paired with a complementary cDNA(8 + 8), which was called one-to-one combination, while MB(15 + 8) was two-to-two combination and MB(15 + 6) was one-to-two combination. When the ratio of SS and cDNA (15 + 8) was 1:1, the quenching efficiency reached maximum. But with the molar ratio of SS and cDNA(8 + 8) increasing, the quenching efficiency increased continuously. Under the optimal conditions that we studied, the detection limit of thrombin by MB(8 + 8) and MB(15 + 8) was 0.19 and 1.2 nM, respectively. In addition, the assay proved to be selective, and the average recovery of thrombin detected by MB(8 + 8) and MB(15 + 8) in diluted serum was 95.4 and 94.5%, respectively.