High-affinity DNA Research Articles

Platinum(II)-Salphen Complexes as DNA Binders and Photosensitisers.

Current anticancer therapies suffer from issues such as off-target side effects and the emergence of drug resistance; therefore, the discovery of alternative therapeutic approaches is vital. These can include the development of drugs with different modes of action, and the exploration of new biomolecular targets. For the former, there has been increasing interest in drugs that are activated by an external stimulus (e. g. light irradiation) to generate cytotoxic chemicals such as reactive oxygen species (ROS). For the latter, significant efforts are being directed to explore non-canonical DNA and RNA structures (e. g. guanine-quadruplexes), as alternative biomolecular targets. Herein we report the synthesis of a library of 21 new platinum(II)-Salphen complexes (square planar platinum(II) complexes coordinated to tetradentate O,N,N,O-Schiff base ligands), and the investigation, for all complexes, of their photophysical and photochemical properties, their interactions with duplex and quadruplex DNA, and their cytotoxicity against HeLa cancer cells both in the dark and upon light irradiation. Thanks to the intrinsic phosphorescence of the platinum(II) complexes, confocal microscopy was used for six of the complexes to determine their cellular permeability and localisation in two cancer cell lines (HeLa and U2OS). Altogether, these studies have allowed us to identify two lead platinum(II) complexes with high guanine-quadruplex DNA affinity and selectivity, good cell permeability and nuclear localisation, and high cytotoxicity against HeLa cancer cells upon irradiation with no detected cytotoxicity in the dark.

Thieno[3,2-b]thiophene for the Construction of Far-Red Molecular Rotor Hemicyanines as High-Affinity DNA Aptamer Fluorogenic Reporters.

The construction of far-red fluorescent molecular rotors (FMRs) is an imperative task for developing nucleic acid stains that have superior compatibility with cellular systems and complex matrices. A typical strategy relies on the methine extension of asymmetric cyanines, which unfortunately fails to produce sensitive rotor character. To break free from this paradigm, we have synthesized far-red hemicyanines using a dimethylamino thieno[3,2-b]thiophene donor. The resultant probes, designated as ATh2Ind and ATh2Btz, possess excitation maxima (λmax) of >600 nm and have been rigorously characterized by NMR, electrochemistry, and computational methods. The dyes possess alternating charge patterns like indodicarbocyanine (Cy5), but with twisted intramolecular charge transfer (TICT) rotational barriers at 60°, akin to the classical FMR thiazole orange (TO1). ATh2Btz also displays cyanine characteristics, enhancing its response upon binding to nucleic acids and allowing for efficient staining of cellular nuclei. When binding to the DNA aptamer for quinine (MN4), ATh2Btz exhibits a Kd of 17 nM, a 660-fold light-up response, brightness (Φfl x εmax) of ∼37,000 M-1cm-1, and λex/λem of 655/677 nm. The resulting far-red DNA-based MN4-ATh2Btz platform has been termed "pomegranate."

Structural basis of nucleic acid recognition by the N-terminal cold shock domain of the plant glycine-rich protein AtGRP2

AtGRP2 is a glycine-rich, RNA-binding protein that plays pivotal roles in abiotic stress response and flowering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal CCHC-type zinc knuckles interspersed with glycine-rich regions. Here, we investigated the structure, dynamics, and nucleic acid binding properties of AtGRP2-CSD. The 2D [1H,15N] HSQC spectrum of AtGRP2-CSD1-79 revealed the presence of a partially folded intermediate in equilibrium with the folded state. The addition of eleven residues at the C-terminus stabilized the folded conformation. The three-dimensional structure of AtGRP2-CSD1-90 unveiled a β-barrel composed of five antiparallel β-strands and a 310 helical turn, along with an ordered C-terminal extension, a conserved feature in eukaryotic CSDs. Direct contacts between the C-terminal extension and the β3-β4 loop further stabilized the CSD fold. AtGRP2-CSD1-90 exhibited nucleic acid binding via solvent-exposed residues on strands β2 and β3, as well as the β3-β4 loop, with higher affinity for DNA over RNA, particularly favoring pyrimidine-rich sequences. Furthermore, DNA binding induced rigidity in the β3-β4 loop, evidenced by 15N-{1H} NOE values. Mutation of residues W17, F26, and F37, in the central β-sheet, completely abolished DNA binding, highlighting the significance of π-stacking interactions in the binding mechanism. These results shed light on the mechanism of nucleic acid recognition employed by AtGRP2, creating a framework for the development of biotechnological strategies aimed at enhancing plant resistance to abiotic stresses.

Palladium–iridium nanocubes modified with a high-affinity DNA aptamer as paired viral diagnostic and therapeutic tools

The COVID-19 pandemic emphasizes the need for the development of molecular tools that can be used as effective diagnostic and therapeutic agents. Herein we investigate the potential of aptamer-dressed nanomaterials both as diagnostics and therapeutics using SARS-CoV-2 as a model. The nanomaterials are based on the palladium-iridium (Pd–Ir) nanocubes modified with monomeric, dimeric or trimeric aptamers that exhibit varying affinities for the spike protein of SARS-CoV-2. These nanomaterials were first examined for diagnostic potential through the creation of a nanozyme-linked aptamer assay (NLAA) that takes advantage of the peroxidase-mimicking activity of Pd–Ir nanocubes. The trimeric aptamer-based NLAA demonstrated a limit of detection (LOD) of 9.3 ​× ​103 cp/mL for pseudoviruses expressing the spike protein of SARS-CoV-2, 172- and 12.9-fold lower than that of the monomeric and dimeric aptamer-based NLAAs, respectively. Upon testing with 60 clinical saliva samples, the trimeric aptamer-based NLAA achieved a specificity of 100 ​% and a sensitivity of 86.7 ​%. The same nanomaterials were also examined for the ability to block viral entry to host cells. The trimeric aptamer-conjugated nanocubes exhibited a superior neutralizing ability, with an IC50 value of 6.4 pM, 2.7- and 10.1-fold lower than that of the dimeric and monomeric aptamer nanocubes. Moreover, the trimeric aptamer-conjugated nanocubes exhibited excellent biostability and biocompatibility. Overall, our study provides a framework for combating future viral pandemics through the development of a paired biosensor and neutralizing agent made of the same aptamer-modified nanomaterial that recognizes an important viral surface protein like the spike protein of SARS-CoV-2.

Selection and Characterization of a DNA Aptamer Recognizing High Mobility Group Box 1 Protein (HMGB1) and Enhancing Its Pro-inflammatory Activity

Background: High mobility group box 1 (HMGB1) plays an essential role in various pathological conditions, including inflammation, fibrosis, autoimmune diseases, and carcinogenesis. The quantification of HMGB1 in body fluids holds promise for clinical applications. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers that target HMGB1. Methods: In this study, ssDNA aptamers were selected using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The affinity and specificity of the aptamers were evaluated through South-Western blot analysis, enzyme-linked aptamer sorbent assay (ELASA), and aptamer-based histochemistry staining. The impact of the aptamers on the biological activity of HMGB1 was tested in the human acute monocytic leukemia cell line, THP-1. Results: An aptamer (H-ap25, dissociation constant = 8.20 ± 0.53 nmol/L) with high affinity for the HMGB1 B box was generated. Further experiments verified that H-ap25 can be used to detect HMGB1 in South-Western blot analysis, ELASA, and aptamer-based histochemistry staining. Moreover, H-ap25 significantly augmented HMGB1-induced expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, Toll-like receptor 9 (TLR9), and activation of NF-κB in THP-1 cells. Conclusions: Our results demonstrated that H-ap25 can be used both as an enhancer of HMGB1 and as a probe in research.

Bioluminescent aptamer-based microassay for detection of melanoma inhibitory activity protein (MIA).

Melanoma inhibitory activity protein (MIA) does obviously offer the potential to reveal clinical manifestations of melanoma. Despite a pressing need for effective diagnosis of this highly fatal disease, there are no clinically approved MIA detection ELISA kits available. A recommended MIA threshold has not yet been defined, mostly by reason of variability in immunoglobulins' affinity and stability, the difference in sample preparation and assay conditions. Here we present a pair of high-affinity DNA aptamers developed as an alternative recognition and binding element for MIA detection. Their stability and reproducible synthesis are expected to ensure this analysis under standard conditions. The devised aptamer-based solid-phase microassay of model standard and control human sera involves luciferase NLuc as a highly sensitive reporter. Bioluminescence dependence on MIA concentration ranges in a linear manner from 2.5 to 250 ng mL-1, providing a MIA detection limit of 1.67 ± 0.57 ng mL-1.

Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Molecular modeling and simulation studies of SELEX-derived high-affinity DNA aptamers to the Ebola virus nucleoprotein

Ebola viral disease (EVD) is a highly infectious and potentially fatal illness with a case fatality rate ranging from 25% to 90%. To effectively control its spread, there is a need for rapid, reliable and lowcost point-of-care (P OC) diagnostic tests. While various EVD diagnostic tests exist, few are P OC tests, and many are not cost-effective. The use of antibodies in these tests has limitations, prompting the exploration of aptamers as potential alternatives. Various proteins from the Ebola virus (EBOV) proteome, including EBOV nucleoprotein (NP), are considered viable targets for diagnostic assays. A previous study identified three aptamers (Apt1. Apt2 and Apt3) with high affinity for EBOV NP using systemic evolution of ligands by exponential enrichment (SELEX). This study aimed to employ in silico methods, such as Phyre2, RNAfold, RNAComposer, HADDOCK and GROMACS, to model the structures of EBOV NP and the aptamers, and to investigate their binding. The in silico analysis revealed successful binding of all the three aptamers to EBOV NP, with a suggested ranking of Apt1 > Apt2 > Apt3 based on binding affinity. Microscale thermophoresis (MST) analysis confirmed the binding, providing dissociation constants of 25 ± 2.84, 56 ± 2.76 and 140 ±3.69 nM for Apt1, Apt2 and Apt3, respectively. The study shows that the findings of the in silico analysis was in agreement with the MST analysis. Inclusion of these in silico approaches in diagnostic assay development can expedite the selection of candidate aptamers, potentially overcoming challenges associated with aptamer application in diagnostics. Communicated by Ramaswamy H. Sarma

A high affinity and selective DNA aptamer for copper ions.

Capture-SELEX was employed for the selection of DNA aptamers for Cu2+. The best aptamer named Cu-1 has a Kd of 14.2 nM as determined using the strand-displacement assay, representing an approximate 3000-fold improvement over a previously reported Cu2+ aptamer. The sensor achieved a limit of detection of 2.1 nM.

Isolation and Identification of the High-Affinity DNA Aptamer Target to the Brain-Derived Neurotrophic Factor (BDNF).

Aptamers are functional oligonucleotide ligands used for the molecular recognition of various targets. The natural characteristics of aptamers make them an excellent alternative to antibodies in diagnostics, therapeutics, and biosensing. DNA aptamers are mainly single-stranded oligonucleotides (ssDNA) that possess a definite binding to targets. However, the application of aptamers to the fields of brain health and neurodegenerative diseases has been limited to date. Herein, a DNA aptamer against the brain-derived neurotrophic factor (BDNF) protein was obtained by in vitro selection. BDNF is a potential biomarker of brain health and neurodegenerative diseases and has functions in the synaptic plasticity and survival of neurons. We identified eight aptamers that have binding affinity for BDNF from a 50-nucleotide library. Among these aptamers, NV_B12 showed the highest sensitivity and selectivity for detecting BDNF. In an aptamer-linked immobilized sorbent assay (ALISA), the NV_B12 aptamer strongly bound to BDNF protein, in a dose-dependent manner. The dissociation constant (Kd) for NV_B12 was 0.5 nM (95% CI: 0.4-0.6 nM). These findings suggest that BDNF-specific aptamers could be used as an alternative to antibodies in diagnostic and detection assays for BDNF.

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for ‎Detection of Epsilon Toxin.

Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies. This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX. This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX. Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 μg/mL for ETX3 and ETX11, respectively.‎. The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.

DNA Aptamer Binding Octapeptide Repeat Region of Cellular Prion Protein.

Cellular prion protein (PrPC) is highly expressed in a variety of tumor cells and plays a crucial role in neurodegenerative diseases. Its N-terminal domain contains a conserved octapeptide (PHGGGWGQ) repeat sequence. The number of repeats has been correlated with the species as well as the development of associated diseases. Herein, PrPC was identified to be the molecular target of a high-affinity DNA aptamer HA5-68 obtained by cell-SELEX. Aptamer HA5-68 was further optimized to two short sequences (HA5-40-1 and HA5-40-2), and its binding site to PrPC was identified to be located in the loop-stem-loop region of the head of its secondary structure. HA5 series aptamers were demonstrated to bind the octapeptide repeat region of PrPC, as well as the synthesized peptides containing different numbers of octapeptide repeats. The PrPC expression on 42 cell lines was measured by using aptamer HA5-68 as a molecular probe. The clear understanding of the molecular structure and binding mechanism of this set of aptamers will provide information for the design of diagnostic methods and therapeutic drugs targeting PrPC.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Self-Assembly Nanostructure Induced by Regulation of G-Quadruplex DNA Topology via a Reduction-Sensitive Azobenzene Ligand on Cells.

The control of DNA assembly systems on cells has increasingly shown great importance for precisely targeted therapies. Here, we report a controllable DNA self-assembly system based on the regulation of G-quadruplex DNA topology by a reduction-sensitive azobenzene ligand. Specifically, three azobenzene multiamines are developed, and AzoDiTren is identified as the best G4 binder, which displays high affinity and specificity for G4 DNA. Moreover, the reduction-sensitive nature of the azobenzene scaffold allows AzoDiTren to induce a complete change of the G4 topology in a tissue-specific manner, even at high metal cation concentrations. On this basis, the AzoDiTren-induced G4 conformational switch achieves control of the self-assembly of G4-functionalized DNAs on cells. This strategy enables the regulation of G4 and DNA self-assembly by the bioreductant-responsive ligand.

Furan-modified PNA probes for covalent targeting and ligation of nucleic acids.

While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic degradation, potential immunogenicity, and delivery issues, which can limit their applications. Peptide Nucleic Acids (PNAs) are promising alternatives due to their high affinity for DNA and RNA, the high resistance to enzymatic degradation, and the easy introduction of a wide range of potential modifications. Chemical modifications that enable the covalent targeting of specific DNA and RNA strands offer additional advantages, including enhanced potency. The current study focuses on the utilization of furan-PNAs as pro-reactive probe systems and their applications to DNA and RNA targeting. Specifically, in this methodological paper, we provide practical insights into the design, synthesis, and application of furan-containing PNA probes for achieving efficient PNA-DNA and PNA-RNA interstrand crosslinking (ICL), as well as ON-templated PNA-PNA ligation systems. Furthermore, we discuss the applications of these probes in targeting DNA secondary structures, such as G-quadruplexes and i-motifs, target pull-down assays, and on-surface detection.

What DNA Sequence Strongly Binds to Carbon Nanotube? High-Throughput Study

Noncovalent functionalization of single-walled carbon nanotubes (SWCNT) with amphiphilic polymers has significantly advanced the colloidal SWCNT efficacy for biomedical optical sensors. Single-strand DNA (ssDNA), as an amphiphilic biocompatible polymer, has been employed to fabricate ssDNA-wrapped SWCNT complexes, however the correlation between DNA sequence and wrapping affinity is not fully studied. In this research, we evolved and screened high-affinity ssDNA sequences from ~1015 ssDNA (random 30-mer), and analyzed the influence of DNA sequence on the wrapping affinity. Wrapping affinity of specific ssDNA sequence was quantitatively measured by the rate constant when ssDNA at the surface was exchanged to sodium cholate. The wrapping affinity relies on the base composition and k-mer motif. Also, we build a machine learning model to identify the high affinity DNA sequence with >85% accuracy. These results indicate the presence of “sequence motif(s)” to control the wrapping affinity and chemical conformation of ssDNA molecule on the SWCNT surface, which can be useful for developing DNA-assisted carbon nanosensors.

Versatile dual-channel loop-mediated isothermal amplification assay featuring smartphone imaging enables determination of fecal indicator bacteria in environmental waters

Rapid diagnostic assays are often a critical tool for monitoring water quality in developing and developed countries. Conventional testing requires 24–48 h for incubation, resulting in delayed remediation and increasing the likelihood of negative outcomes. In this study, we report a workflow for detection of E. coli, a common indicator of fecal contamination. Following large volume filtration, E. coli is then solubilized enabling the facile isolation and recovery of genetic material by a thin film microextraction (TFME) device featuring a polymeric ionic liquid (PIL) sorbent. Rapid recovery of pure nucleic acids is achieved using a PIL sorbent with high affinity for DNA to significantly increase mass transfer and facilitate adsorption and desorption of DNA. Downstream detection is performed by a versatile, dual channel loop mediated isothermal amplification (LAMP) assay featuring a colorimetric dye and a sequence-specific molecular beacon. A portable LAMP companion box enables consistent isothermal heating and endpoint smartphone imaging while being powered by a single 12-V battery. Programmable LEDs are switched from white or blue light to facilitate the independent imaging of the colorimetric dye or fluorometric probe following amplification. The methodology positively identified E. coli in environmental samples spiked to concentrations of 6600 colony forming units (CFU) per milliliter and 660 CFU/mL with 100% and 22% positivity, respectively.

Development of a Repair Enzyme Fluorescent Probe to Reveal the Intracellular DNA Damage Induced by Benzo[a]pyrene in Living Cells

Pollutant exposure causes a series of DNA damage in cells, resulting in the initiation and progression of diseases and even cancers. An investigation of the DNA damage induced by pollutants in living cells is significant to evaluate the cytotoxicity, genotoxicity, and carcinogenicity of environmental exposure, providing critical insight in the exploration of the etiologies of diseases. In this study, we develop a repair enzyme fluorescent probe to reveal the DNA damage caused by an environmental pollutant in living cells by single-cell fluorescent imaging of the most common base damage repair enzyme named human apurinic/apyrimidinic endonuclease 1 (APE1). The repair enzyme fluorescent probe is fabricated by conjugation of an APE1 high affinity DNA substrate on a ZnO2 nanoparticle surface to form a ZnO2@DNA nanoprobe. The ZnO2 nanoparticle serves as both a probe carrier and a cofactor supplier, releasing Zn2+ to activate APE1 generated by pollutant exposure. The AP-site in the DNA substrate of the fluorescent probe is cleaved by the activated APE1, releasing fluorophore and generating fluorescent signals to indicate the position and degree of APE1-related DNA base damage in living cells. Subsequently, the developed ZnO2@DNA fluorescent probe is applied to investigate the APE1-related DNA base damage induced by benzo[a]pyrene (BaP) in living human hepatocytes. Significant DNA base damage by BaP exposure is revealed, with a positive correlation of the damage degree with exposure time in 2-24 h and the concentration in 5-150 μM, respectively. The experimental results demonstrate that BaP has a significant effect on the AP-site damage, and the degree of DNA base damage is time-dependent and concentration-dependent.

Generation of a high-affinity DNA aptamer for on-site screening of toxic aristolochic acid I in herbal medicines and botanical products

Aristolochic Acid I (AAI) is an environmental and foodborne toxin found in the Aristolochia and Asarum species of plants that are widespread all over the world. Therefore, there is an urgent need to develop a sensitive and specific biosensor for identifying AAI. Aptamers as a powerful biorecognition element provide the most viable options for solving this problem. In this study, we used library-immobilized SELEX to isolate an AAI-specific aptamer with a KD value of 86 ± 13 nM. To verify the practicability of the selected aptamer, a label-free colorimetric aptasensor was designed. This aptasensor exhibited a low detection limit of 225 nM. Besides, it had been further applied for the determination of AAI in real samples and the recoveries ranged from 97.9% to 102.4%. In the future, AAI aptamer will provide a promising tool for safety evaluation in various fields of agriculture, food, and medication.

Large SYBR Green I fluorescence enhancement for label-free aptamer-based detection of estradiol

Estradiol (E2) and related estrogens are emerging environmental contaminants that may adversely affect the health of humans, animals, and ecosystems. Many aptamers have been reported for the detection of E2, and our lab recently selected a series of high-affinity and short DNA aptamers that showed various binding orientations to E2, leading to different selectivity patterns. In this work, we report that using SYBR Green I (SGI) as a fluorescence probe, up to 200% fluorescence increase was achieved upon titration of E2 to these aptamers. Such enhancement was the highest among all reported small molecule binding aptamers using SGI for signal generation, although some metal-binding DNA can achieve even higher enhancement. By gradually shortening the stem region of an E2 binding aptamer, we concluded that the enhanced fluorescence was from the aptamer binding pocket upon target binding instead of from the duplexed stem region. Comparison was also made with a few other aptamers including those for caffeine, quinine, uric acid and cortisol, and none of them showed more than 20% fluorescence change. Using the SGI method, the detection limit was calculated to be 2.4 nM E2. We attributed the large fluorescence enhancement to the hydrophobic nature of E2 and the high-affinity binding of the aptamers. This study provides insights into the aptamers that can use SGI for their binding assays and biosensor development.