Systematic Evolution Of Ligands Research Articles

Truncations and in silico Docking to Enhance the Analytical Response of Aptamer-Based Biosensors

Aptamers are short oligonucleotides capable of binding specifically to various targets (i.e., small molecules, proteins, and whole cells) which have been introduced in biosensors such as in the electrochemical aptamer-based (E-AB) sensing platform. E-AB sensors are comprised of a redox-reporter-modified aptamer attached to an electrode that undergoes, upon target addition, a binding-induced change in electron transfer rates. To date, E-AB sensors have faced a limitation in the translatability of aptamers into the sensing platform presumably because sequences obtained from Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are typically long (> 80 nucleotides) and that obtaining structural information remains time and resource consuming. In response, we explore the utility of aptamer base truncations and in silico docking to improve their translatability into E-AB sensors. Here, we first apply this to the glucose aptamer, which we characterize in solution using NMR methods to guide design and translate truncated variants in E-AB biosensors. We further investigated the applicability of the truncation and computational approaches to four other aptamer systems (vancomycin, cocaine, methotrexate and theophylline) from which we derived functional E-AB sensors. We foresee that our strategy will increase the success rate of translating aptamers into sensing platforms to afford low-cost measurements of molecules directly in undiluted complex matrices.

Isolation and Characterization of ssDNA Aptamers against BipD antigen of Burkholderia pseudomallei

BackgroundMelioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD. MethodsThe recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay. ResultsThe fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria. ConclusionAptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.

Establishment of the rapid electrochemical-SELEX monitoring system by using ACEF technique and its application to dengue virus aptasensor fabrication

Aptamers, which consist of single-stranded DNA or RNA, possess considerable potential as biomolecular recognition elements. The systematic evolution of ligands by exponential enrichment (SELEX) serves as a general method for synthesizing aptamers. However, the conventional SELEX process for monitoring aptamer-target binding is time-consuming and labor-intensive, highlighting the need for rapid monitoring and selection methods. To address this problem, this study introduces a novel approach called rapid electrochemical-SELEX (RE-SELEX) monitoring method, which significantly reduces the target-aptamer binding time from 3 h to 10 min. This process is achieved by employing an alternating current electrothermal flow (ACEF) technique. The Au micro gap-type electrode was used to RE-SELEX monitoring device. As a proof-concept of this method, the dengue virus (DENV) aptamers were synthesized. For confirming the feasibility of this DENV aptamer, we analyzed target binding of produced aptamers test including gel electrophoresis, dissociation constant analysis. Then, two aptamers were selected for constructing the rapid electrochemical DENV aptasensor. The RE biosensor was achieved by ACEF technique that reduce the target-aptamer binding within 10 min. The fabricated device can detect 37 fM of the target dengue envelope protein in diluted human serum well. Thus, the proposed method provides a reliable SELEX-monitoring method for the rapid synthesis of aptamers using a simple electrochemical platform.

Screening biotoxin aptamer and their application of optical aptasensor in food stuff: a review.

Biotoxins are ranges of toxic substances produced by animals, plants, and microorganisms, which could contaminate foods during their production, processing, transportation, or storage, thus leading to foodborne illness, even food terrorism. Therefore, proposing simple, rapid, and effective detection methods for ensuring food free from biotoxin contamination shows a highly realistic demand. Aptamers are single-stranded oligonucleotides obtained from the systematic evolution of ligands by performing exponential enrichment (SELEX). They can specifically bind to wide ranges of targets with high affinity; thus, they have become important recognizing units in safety monitoring in food control and anti-terrorism. In this paper, we reviewed the technical points and difficulties of typical aptamer screening processes for biotoxins. For promoting the understanding of food control in the food supply chain, the latest progresses in rapid optical detection of biotoxins based on aptamers were summarized. In the end, we outlined some challenges and prospects in this field. We hope this paper could stimulate widespread interest in developing advanced sensing systems for ensuring food safety.

Aptamer Screening: Current Methods and Future Trend towards Non-SELEX Approach.

Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide non-SELEX methods into an immobilized format and non-immobilized format and discuss how high-resolution partitioning methods could facilitate the further improvement of selection efficiency and accuracy.

Breaking Barriers: Nucleic Acid Aptamers in Gastrointestinal (GI) Cancers Therapy.

Conventional cancer therapies can have significant adverse effects as they are not targeted to cancer cells and may damage healthy cells. Single-stranded oligonucleotides assembled in a particular architecture, known as aptamers, enable them to attach selectively to target areas. Usually, they are created by Systematic Evolution of Ligand by Exponential enrichment (SELEX), and they go through a rigorous pharmacological revision process to change their therapeutic half-life, affinity, and specificity. They could thus offer a viable substitute for antibodies in the targeted cancer treatment market. Although aptamers can be a better choice in some situations, antibodies are still appropriate for many other uses. The technique of delivering aptamers is simple and reasonable, and the time needed to manufacture them is relatively brief. Aptamers do not require animals or an immune response to be produced, in contrast to antibodies. When used as a medication, aptamers can directly suppress tumor cells. As an alternative, they can be included in systems for targeted drug delivery that administer medications specifically to tumor cells while reducing toxicity to healthy cells. The most recent and cutting-edge methods for treating gastrointestinal (GI) tract cancer with aptamers will be covered in this review, with a focus on targeted therapy as a means of conquering resistance to traditional medicines.

Detection of dual-methylated BRCA1/BRCA2 cell-free DNA for ovarian cancer on an aptamer-based integrated microfluidic system

A new screening strategy has been developed to select nucleic acid aptamer probes with an “in vivo-like” systematic evolution of ligands by exponential enrichment (SELEX) process featuring positive, negative selection and in vivo-like selection steps. One methylation-specific aptamer with high affinity and specificity was selected and it could capture methylated cell-free DNA (cfDNA) from unfiltered blood of ovarian cancer (OvCa) patients. This aptamer was conjugated onto magnetic beads and integrated into a new microfluidic chip for rapid and automatic detection and quantification of two prevalent methylated tumor suppressor genes, breast cancer type 1 and 2 susceptibility protein (BRCA 1/2), in the molecular profile of OvCa. The assay was conducted under physiological conditions that mimic the microenvironment of cancer patients at 25–37 °C, with a low pH value (5.5) and/or high blood ion levels, without requiring additional sample preparation steps. The results of this study could be extrapolated to the direct detection of methylated cfDNA at levels as low as 1 pM in blood or plasma. Not only was DNA methylation observed in OvCa patients, but it was also detected in many other types of cancer; it is therefore considered an important factor in carcinogenesis. Hence, it is inferred that this screened aptamer should not only be used for OvCa diagnosis but also for multiple cancer epigenetic detection and drug development research in the future.

Topical application of a CCL22-binding aptamer suppresses contact allergy

Allergic contact dermatitis is a prevalent occupational disease with limited therapeutic options. The chemokine CCL22, a ligand of the chemokine receptor CCR4, directs the migration of immune cells. Here, it is shown that genetic deficiency of CCL22 effectively ameliorated allergic reactions in contact hypersensitivity (CHS), a commonly used mouse model of allergic contact dermatitis. For pharmacological inhibition of CCL22, DNA aptamers specific for murine CCL22 were generated by systematic evolution of ligands by exponential enrichment (SELEX). Nine CCL22-binding aptamers were initially selected and functionally tested in vitro. The 29-nucleotide DNA aptamer AJ102.29m profoundly inhibited CCL22-dependent T cell migration and did not elicit undesired Toll-like receptor-dependent immune activation. AJ102.29m efficiently ameliorated CHS in vivo after systemic application. Moreover, CHS-associated allergic symptoms were also reduced following topical application of the aptamer on the skin. Microscopic analysis of skin treated with AJ102.29m ex vivo demonstrated that the aptamer could penetrate into the epidermis and dermis. The finding that epicutaneous application of the aptamer AJ102.29m in a cream was as effective in suppressing the allergic reaction as intraperitoneal injection paves the way for therapeutic use of aptamers beyond the current routes of systemic administration.

Development of Aptamers for RNase Inactivation in Xtract-Free™ Sample Collection and Transport Medium.

There is a significant need to develop new environmentally friendly, extraction-free sample collection mediums that can effectively preserve and protect genetic material for point-of-care and/or self-collection, home-collection, and mail-back testing. Systematic evolution of ligands by exponential enrichment (SELEX) was used to create anti-ribonuclease (RNase) deoxyribonucleic acid (DNA) aptamers against purified RNase A conjugated to paramagnetic carboxylated beads. Following eight rounds of SELEX carried out under various stringency conditions, e.g., selection using Xtract-Free™ (XF) specimen collection medium and elevated ambient temperature of 28 °C, a panel of five aptamers was chosen following bioinformatic analysis using next-generation sequencing. The efficacy of aptamer inactivation of RNase was assessed by monitoring ribonucleic acid (RNA) integrity via fluorometric and real-time RT-PCR analysis. Inclusion of aptamers in reaction incubations resulted in an 8800- to 11,200-fold reduction in RNase activity, i.e., digestion of viral RNA compared to control. Thus, anti-RNase aptamers integrated into XF collection medium as well as other commercial reagents and kits have great potential for ensuring quality intact RNA for subsequent genomic analyses.

Selection of a new aptamer targeting amoxicillin for utilization in a label-free electrochemical biosensor

Pharmaceutical pollution has received considerable attention because of the harmful effects of pharmaceutical compounds on human health, even in trace amounts. Amoxicillin is one of the frequently used antibiotics that was included in the list of emerging water pollutants. Therefore, a highly selective and rapid technique for amoxicillin detection is required. In this work, a new aptamer was selected for amoxicillin and utilized for the development of a label-free electrochemical aptasensor. Aptamer selection was performed using the systematic evolution of ligands by exponential enrichment. The selected aptamer showed good specificity against other antibiotics, including the structurally related antibiotics: ampicillin and ciprofloxacin. Among the selected aptamers, Amx3 exhibited the lowest dissociation constant value of 112.9 nM. An aptasensor was developed by immobilization of thiolated Amx3 aptamer onto gold screen–printed electrodes via self-assembly, which was characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The detection was realized by monitoring the change in the differential pulse voltammetry peak current in the ferro/ferricyanide redox couple upon binding of the aptasensor to amoxicillin. The aptasensor showed very good sensitivity with an ultralow limit of detection of 0.097 nM. When the aptasensor was tested using actual spiked milk samples, excellent recovery percentages were observed. The label-free electrochemical aptasensor developed herein is a promising tool for the selective and sensitive detection of amoxicillin in environmental samples.

Unveiling the Role of RNA Recognition Motif Proteins in Orchestrating Nucleotide-Binding Site and Leucine-Rich Repeat Protein Gene Pairs and Chloroplast Immunity Pathways: Insights into Plant Defense Mechanisms.

In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.

Antidote-controlled DNA aptamer modulates human factor IXa activity

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

In vitro selection of DNA aptamers against staphylococcal enterotoxin A

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Designing molecules: directing stem cell differentiation.

Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

Screening of Cross-Reactive Aptamers for the Detection of 24 Quinolones by Using a Liebig's Law-Guided Parallel-Series Strategy.

Quinolones, a widely used class of antibiotics, present significant environmental and health concerns if they excessively remain in the environment and in food. Aptamers specific to quinolones can be applied as bioreceptors for the detection of quinolone residues in the environment and food. The quinolone family contains dozens of different individuals that share the same core structure coupled with various substituents at six different positions. The diversity and complexity of the substitution sites make it a challenge to choose a set of representative molecules that encompass all the desired sites and preserve the core molecular framework for the screening of quinolone-specific aptamers via systematic evolution of ligands by exponential enrichment (SELEX). To address this challenge, we introduce a novel parallel-series strategy guided by Liebig's law for isolating quinolone-specific cross-reactive aptamers by using the library-immobilized SELEX method. Through this approach, we successfully identified 5 aptamers (Apt.AQ01-Apt.AQ05) with high binding affinity and excellent specificity to 24 different quinolone individuals. Among them, Apt.AQ03 showcased optimal performance with affinities ranging from 0.14 to 1.07 μM across the comprehensive set of 24 quinolones, exhibiting excellent specificity against nontarget interferents. The binding performance of Apt.AQ03 was further characterized with microscale thermophoresis, circular dichroism spectra, and an exonuclease digestion assay. By using Apt.AQ03 as a bioreceptor, a fluorescence resonance energy transfer (FRET) aptasensor was developed for the detection of 24 quinolones in milk, achieving a remarkable detection limit of 14.5-21.8 ng/mL. This work not only establishes a robust and effective strategy for selecting cross-reactive aptamers applicable to other small-molecule families but also provides high-quality aptamers for developing various high-throughput and reliable methods for the detection of multiple quinolone residues in food.

Aptamer-modified Nanocarrier(s) for the Management of Prostate Cancer

Abstract: Prostate cancer remains a significant global health concern, necessitating the development of innovative therapeutic approaches. This editorial provides an overview of the current state of research on aptamer-modified nanocarriers as a novel strategy for prostate cancer therapy. Aptamers, known for their high specificity and affinity, have drawn much attention in both research and the pharmaceutical industry. The Systematic Evolution of Ligands by the Exponential Enrichment (SELEX) technique has enabled the development of aptamers that can selectively bind to target ligands based on their unique three-dimensional structures. Notably, the A10 aptamer, modified to target PSMA (Prostate-Specific Membrane Antigen), has demonstrated strong binding affinity and specificity to prostate cancer cells, offering promise as an efficient drug-delivery system. Additionally, biosensors, like the innovative apta-sensor targeting MUC1, have utilized aptamers to detect cancer through electrochemical signals. Additionally, aptamers have improved the pharmacological attributes of chemotherapeutic agents for prostate cancer treatment. Aptamer-- mediated drug delivery has offered enhanced drug delivery, targeting, cellular uptake, and minimizing side effects.

Development of a CRISPR/Cas12a-mediated aptasensor for Mpox virus antigen detection

The emergence and rapid spread of Mpox (formerly monkeypox) have caused significant societal challenges. Adequate and appropriate diagnostics procedures are an urgent necessity. Herein, we discover a pair of aptamers through the systematic evolution of ligands by exponential enrichment (SELEX) that exhibit high affinity and bind to different sites towards the A29 protein of the Mpox virus. Subsequently, we propose a facile, sensitive, convenient CRISPR/Cas12a-mediated aptasensor for detecting the A29 antigen. The procedure employs the bivalent aptamers recognition, which induces the formation of a proximity switch probe and initiates subsequent cascade strand displacement reactions, then triggers CRISPR/Cas12a DNA trans-cleavage to achieve the sensitive detection of Mpox. Our method enables selective and ultrasensitive evaluation of the A29 protein within the range of 1 ng mL−1 to 1 μg mL−1, with a limit of detection (LOD) at 0.28 ng mL−1. Moreover, spiked A29 protein recovery exceeds 96.9%, while the detection activity remains above 91.9% after six months of storage at 4 °C. This aptasensor provides a novel avenue for exploring clinical diagnosis in cases involving Mpox as facilitating development in various analyte sensors.

Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.

Advancing the Diagnosis of Periprosthetic Joint Infections: Integrated Microfluidic Platform for Alpha-Defensins-Specific Aptamer Selection and Its Analytical Applications.

Periprosthetic joint infections (PJIs) pose a significant challenge in orthopedic surgery, particularly total joint arthroplasty (TJA), due to the potential for implant failure and increased patient morbidity. Early and accurate detection of PJIs is crucial for timely intervention and better patient prognosis. Herein, we successfully screened a high-affinity aptamer targeting alpha-defensin complex human neutrophil protein 1-3 (HNP 1-3; potential PJI biomarkers in synovial fluid [SF]) for the first time using systematic evolution of ligands by exponential enrichment (SELEX) on an integrated microfluidic platform. The compact microfluidic device enabled efficient screening, with each round completed within <2 h, comprising five rounds of positive selection, two rounds of negative selection, and one round of competitive selection. A novel one-aptamer-one-antibody assay was further developed from the optimal aptamer screened, and it could accurately quantify HNP 1-3 in SF within 3 h with only ∼50 μL of SF. The assay demonstrated strong binding affinity and specificity for the target protein in SF. Thirteen PJI SF samples were accurately diagnosed and the assay was accurate over a wide dynamic range (0.32-100 mg/L). This study has showcased a rapid and accurate diagnostic tool for PJI detection, which should see widespread use in the clinic, holding promise for potential analytical applications in orthopedic surgery and improving patient care.

Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.