Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology.

The use of polymerase enzymes in biotechnology has allowed us to gain unprecedented control over the manipulation of DNA, opening up new and exciting applications in areas such as biosensing, polynucleotide synthesis, and DNA storage, aptamer development and DNA-nanotechnology. One of the most intriguing enzymes which has gained prominence in the last decade is terminal deoxynucleotidyl transferase (TdT), which is one of the only polymerase enzymes capable of catalysing the template independent stepwise addition of nucleotides onto an oligonucleotide chain. This unique enzyme has seen a significant increase in a variety of different applications. In this review, we give a comprehensive discussion of the unique properties and applications of TdT as a biotechnology tool, and the application in the enzymatic synthesis of poly/oligonucleotides. Finally, we look at the increasing role of TdT enzyme in biosensing, DNA storage, synthesis of DNA nanostructures and aptamer development, and give a future outlook for this technology.

Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides.

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

The Protein-Templated Synthesis of Enzyme-Generated Aptamers.

Inspired by the chemical synthesis of molecularly imprinted polymers, we demonstrated for the first time, the protein-target mediated synthesis of enzyme-generated aptamers (EGAs). We prepared pre-polymerisation mixtures containing different ratios of nucleotides, an initiator sequence and protein template and incubated each mixture with terminal deoxynucleotidyl transferase (TdT). Upon purification and rebinding of the EGAs against the target, we observed an enhancement in binding of templated-EGAs towards the target compared to a non-templated control. These results demonstrate the presence of two primary mechanisms for the formation of EGAs, namely, the binding of random sequences to the target as observed in systematic evolution of ligands by exponential enrichment (SELEX) and the dynamic competition between TdT enzyme and the target protein for binding of EGAs during synthesis. The latter mechanism serves to increase the stringency of EGA-based screening and represents a new way to develop aptamers that relies on rational design.

DNA aptamers against the DUX4 protein reveal novel therapeutic implications for FSHD.

Aberrant expression of the transcription factor double homeobox protein 4 (DUX4) can lead to a number of diseases including facio-scapulo-humeral muscular dystrophy (FSHD), acute lymphoblastic leukemia, and sarcomas. Inhibition of DUX4 may represent a therapeutic strategy for these diseases. By applying Systematic Evolution of Ligands by EXponential Enrichment (SELEX), we identified aptamers against DUX4 with specific secondary structural elements conveying high affinity to DUX4 as assessed by fluorescence resonance energy transfer and fluorescence polarization techniques. Sequences analysis of these aptamers revealed the presence of two consensus DUX4 motifs in a reverse complementary fashion forming hairpins interspersed with bulge loops at distinct positions that enlarged the binding surface with the DUX4 protein, as determined by crystal structure analysis. We demonstrate that insertion of specific structural elements into transcription factor binding oligonucleotides can enhance specificity and affinity.

The Composition of Reconstituted High-Density Lipoproteins (rHDL) Dictates the Degree of rHDL Cargo- and Size-Remodeling via Direct Interactions with Endogenous Lipoproteins.

The application of reconstituted high-density lipoproteins (rHDL) as a drug-carrier has during the past decade been established as a promising approach for effective receptor-mediated drug delivery, and its ability to target tumors has recently been confirmed in a clinical trial. The rHDL mimics the endogenous HDL, which is known to be highly dynamic and undergo extensive enzyme-mediated remodulations. Hence, to reveal the physiological rHDL stability, a thorough characterization of the dynamics of rHDL in biologically relevant environments is needed. We employ a size-exclusion chromatography (SEC) method to evaluate the dynamics of discoidal rHDL in fetal bovine serum (FBS), where we track both the rHDL lipids (by the fluorescence from lipid-conjugated fluorophores) and apoA-I (by human apoA-I ELISA). We show by using lipoprotein depleted FBS and isolated lipoproteins that rHDL lipids can be transferred to endogenous lipoproteins via direct interactions in a nonenzymatic process, resulting in rHDL compositional- and size-remodeling. This type of dynamics could lead to misinterpretations of fluorescence-based rHDL uptake studies due to desorption of labile lipophilic fluorophores or off-target side effects due to desorption of incorporated drugs. Importantly, we show how the degree of rHDL remodeling can be controlled by the compositional design of the rHDL. Understanding the correlation between the molecular properties of the rHDL constituents and their collective dynamics is essential for improving the rHDL-based drug delivery platform. Taken together, our work highlights the need to carefully consider the compositional design of rHDL and test its stability in a biological relevant environment, when developing rHDL for drug delivery purposes.

Peanut Allergy: Characteristics and Approaches for Mitigation.

Peanut allergy has garnered significant attention because of the high sensitization rate, increase in allergy, and severity of the reaction. Sufficiently reliable therapies and efficient mitigating techniques to combat peanut allergy are still lacking. Current management relies on avoiding peanuts and nuts and seeds with homologous proteins, although adverse events mostly occur with accidental ingestion. There is a need for hypoallergenic peanut products to protect sensitized individuals and perhaps serve as immunotherapeutic products. Alongside traditional practices of thermal and chemical treatment, novel processing approaches such as high-pressure processing, pulsed ultraviolet light, high-intensity ultrasound, irradiation, and pulsed electric field have been performed toward reducing the immunoreactivity of peanut. Covalent and noncovalent chemical modifications to proteins also have the tendency to alter peanut allergenicity. Enzymatic hydrolysis seems to be the most advantageous technique in diminishing the allergenic potential of peanut. Furthermore, the combined processing approach (hurdle technologies) such as enzymatic hydrolysis followed by, or in conjunction with, roasting, high pressure and heat, ultrasound with enzymatic treatment, or germination have shown a significant reduction of peanut immunoreactivity and may emerge as useful techniques in reducing the allergenicity of peanut and other foods. This study represents our current knowledge about the alterations in allergenic properties of peanut via different processing mechanisms as well as evaluating its future potential, geographical based data on increasing sensitization, clinical relevance, eliciting dose, and current management of peanut allergy. Furthermore, the molecular characteristics and clinical relevance of peanut allergens have been discussed.

Molecularly imprinted nanoparticles for inhibiting ribonuclease in reverse transcriptase polymerase chain reaction.

Molecularly imprinted nanoparticles (nanoMIPs) are synthesized via a solid-phase approach using RNase as the template. The feasibility of employing the nanoMIPs as RNase inhibitor is successfully demonstrated in reverse transcriptase polymerase chain reaction (RT-PCR) assays, suggesting the tailor-made nanomaterials are very promising for use in routine biological assays.

Fluorometric determination of doxycycline based on the use of carbon quantum dots incorporated into a molecularly imprinted polymer.

A fluorometric assay is described for doxycycline detection. It is based on the use of nitrogen-doped carbon quantum dots (NCQDs) coated with molecularly imprinted polymers (MIPs). The NCQDs were prepared by a one-step hydrothermal reaction using citric acid and ethylenediamine (EDA) as the starting materials. Afterwards, the NCQDs were incorporated into the polymer that was molecularly imprinted with doxycycline. It is found that doxycycline quenches the fluorescence of the NCQDs, and that the functional groups on the surface of NCQDs play an important role in terms of quenching efficiency. A larger fraction of carboxyl groups presented on the surface of NCQDs leads to a higher quenching efficiency due to the enhanced electron transfer from NCQD to doxycycline. The NCQDs@MIP composite can specifically and rapidly recognize doxycycline. Fluorescence drops linearly in the 5 to 50 µM doxycycline concentration range, and the limit of detection is 87 nM. This method was successfully applied to the determination of doxycycline in spiked pig serum where it gave recovery rates of >94%. Graphical abstract Schematic illustration for fabricating a fluorescent sensor based on nitrogen-doped carbon quantum dots (NCQDs) and molecularly imprinted polymers (MIPs). The sensor integrates the merits of the high sensitivity of NCQD and good selectivity of MIPs, and can be significantly quenched upon interaction with doxycycline.

Quantitative Detection of Trace Level Cloxacillin in Food Samples Using Magnetic Molecularly Imprinted Polymer Extraction and Surface-Enhanced Raman Spectroscopy Nanopillars.

There is an increasing demand for rapid, sensitive, and low cost analytical methods to routinely screen antibiotic residues in food products. Conventional detection of antibiotics involves sample preparation by liquid-liquid or solid-phase extraction, followed by analysis using liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or gas chromatography (GC). The process is labor-intensive, time-consuming, and expensive. In this study, we developed a new analytical method that combines magnetic molecularly imprinted polymer (MMIP)-based sample preparation with surface-enhanced Raman spectroscopy (SERS)-based detection for quantitative analysis of cloxacillin in pig serum. MMIP microspheres were synthesized using a core-shell technique. The large loading capacity and high selectivity of the MMIP microspheres enabled efficient extraction of cloxacillin, while the magnetically susceptible characteristics greatly simplified sample handling procedures. Low cost and robust SERS substrates consisting of vertical gold capped silicon nanopillars were fabricated and employed for the detection of cloxacillin. Quantitative SERS was achieved by normalizing signal intensities using an internal standard. By coherently combining MMIP extraction and silicon nanopillar-based SERS biosensor, good sensitivity toward cloxacillin was achieved. The detection limit was 7.8 pmol. Cloxacillin recoveries from spiked pig plasma samples were found to be more than 80%.

The use of differential scanning fluorimetry in the rational design of plastic antibodies for protein targets.

The development of molecularly imprinted polymer nanoparticles (MIP-NPs), which specifically bind biomolecules, is of great interest in the area of biosensors, sample purification, therapeutic agents and biotechnology. Polymerisation techniques such as precipitation polymerisation, solid phase synthesis and core shell surface imprinting have allowed for significant improvements to be made in developing MIP-NPs which specifically recognise proteins. However, the development of MIP-NPs for protein templates (targets) still require lengthy optimisation and characterisation using different ratios of monomers in order to control their size, binding affinity and specificity. In this work we successfully demonstrated that differential scanning fluorimetry (DSF) can be used to rapidly determine the optimum imprinting conditions and monomer composition required for MIP-NP design and polymerisation. This is based on the stability of the protein template and shift in apparent melting points (Tm) upon interaction with different functional acrylic monomers. The method allows for the characterisation of molecularly imprinted nanoparticles (MIP-NPs) due to the observed differences in melting point profiles between, protein-MIP-NPs complexes, pre-polymerisation mixtures and non-imprinted nanoparticles (NIP-NPs) without the need for prior purification. The technique is simple, rapid and can be carried out on most quantitative polymerase chain reaction (qPCR) thermal cyclers which have the required filters for SYPRO

Selection of cholesterol esterase aptamers using a dual-partitioning approach.

Non-systematic evolution of ligands by exponential enrichment and other capillary-based methods have grown in popularity for selection of aptamers since they provide a fast and efficient partitioning method when compared to classical techniques. Despite promising developments in these techniques, a major obstacle needs to be overcome for capillary-based selections to be widely accepted. Due to the small injection volumes associated with CE, only a small proportion of the nucleic acid library can be partitioned at any one time. In this paper, we propose a new two-step method for the selection of aptamers which firstly incorporates a nitrocellulose membrane filter followed by CE. This technique allows for nonbinding sequences to be eliminated, reducing the library size before subsequent capillary-based partitioning, while still reducing the time taken for aptamers to be selected. We demonstrated this technique on the selection of aptamers for cholesterol esterase and the highest binding truncated aptamer CES 4T displayed a K(D) of 203 ± 14 nM. In addition, an increase in the number of sequences partitioned was estimated using spectrophotometry and capillary injection volumes. The results suggested that for successful selection a two-step approach is needed. This hybrid technique could be used to select aptamers that bind to targets both in solution and immobilized onto a stationary phase, allowing the aptamers to be used in different binding environments.

Three-dimensional selection of leptin aptamers using capillary electrophoresis and implications for clone validation.

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has been used as a fast and efficient way to select aptamers against protein targets and offers the advantage of separating bound DNA from unbound DNA in a free solution three-dimensional environment. CE-SELEX was used to select aptamers against human leptin protein. Two methods used to validate the aptamers' binding affinity against the target were performed and gave differing results. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) yielded K(D) values in the high nanomolar range, whereas the fluorescence intensity method gave K(D) values in the low micromolar range. These results may suggest that aptamer validation must be carried out in a similar environment to that of the selection partitioning step and the environment in which the aptamer is intended to be used. We also note that affinity binding by fluorescence intensity using microplate readers may be limited to targets that have relatively low k(off) rates, systematic errors may occur when aptamers are validated using two different techniques, and the immobilization of smaller targets onto plate wells can affect the binding of the DNA, giving rise to lower binding affinities.

Selection of bovine catalase aptamers using non-SELEX.

In this research, we used the non-SELEX method to successfully select an aptamer that binds to the protein target (bovine catalase) with a K(D) value in the low micro molar range. The time window was determined for the target and aptamer library by optimizing the buffer conditions using 3 × Tris-glycine-potassium phosphate (TGK) buffer as the run buffer and 1× TGK as the selection buffer to give the biggest complex peak. Fractions were collected by multistep nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based partitioning for three rounds of selection. The fractions from each round were enriched using PCR and the progress of selection was monitored using bulk affinity analysis. Fraction 2 was determined to have the optimal bulk affinity (0.75 µM) and this enriched library was cloned and sequenced giving four aptamer sequences. These sequences were verified using affinity capillary electrophoresis (CAT 1 0.237 µM) and fluorescence intensity measurements (CAT 1 0.430 µM). The specificity of the aptamer was also determined by fluorescence intensity measurements. The results showed that the aptamer with the highest binding affinity showed at least a 100-fold decrease in affinity toward four other proteins (i.e. lysozyme, trypsinogen, chymotrypsinogen A, and myoglobin) tested and this confirmed that the aptamer exhibited a distinct specificity toward bovine catalase. This aptamer will be useful in biosensing, Western blot, and biomarker identification.

Anti-MicroRNA-21 Oligonucleotide Loaded Spermine-Modified Acetalated Dextran Nanoparticles for B1 Receptor-Targeted Gene Therapy and Antiangiogenesis Therapy.

The use of nanoparticles (NPs) to deliver small inhibiting microRNAs (miRNAs) has shown great promise for treating cancer. However, constructing a miRNA delivery system that targets brain cancers, such as glioblastoma multiforme (GBM), remains technically challenging due to the existence of the blood-tumor barrier (BTB). In this work, a novel targeted antisense miRNA-21 oligonucleotide (ATMO-21) delivery system is developed for GBM treatment. Bradykinin ligand agonist-decorated spermine-modified acetalated dextran NPs (SpAcDex NPs) could temporarily open the BTB by activating G-protein-coupled receptors that are expressed in tumor blood vessels and tumor cells, which increase transportation to and accumulation in tumor sites. ATMO-21 achieves high loading in the SpAcDex NPs (over 90%) and undergoes gradual controlled release with the degradation of the NPs in acidic lysosomal compartments. This allows for cell apoptosis and inhibition of the expression of vascular endothelial growth factor by downregulating hypoxia-inducible factor (HIF-1α) protein. An in vivo orthotopic U87MG glioma model confirms that the released ATMO-21 shows significant therapeutic efficacy in inhibiting tumor growth and angiogenesis, demonstrating that agonist-modified SpAcDex NPs represent a promising strategy for GBM treatment combining targeted gene therapy and antiangiogenic therapy.

Upregulating Aggregation-Induced-Emission Nanoparticles with Blood-Tumor-Barrier Permeability for Precise Photothermal Eradication of Brain Tumors and Induction of Local Immune Responses.

Compared to other tumors, glioblastoma (GBM) is extremely difficult to treat. Recently, photothermal therapy (PTT) has demonstrated advanced therapeutic efficacy; however, because of the relatively low tissue-penetration efficiency of laser light, its application in deep-seated tumors remains challenging. Herein, bradykinin (BK) aggregation-induced-emission nanoparticles (BK@AIE NPs) are synthesized; these offer selective penetration through the blood-tumor barrier (BTB) and strong absorbance in the near-infrared region (NIR). The BK ligand can prompt BTB adenosine receptor activation, which enhances transportation and accumulation inside tumors, as confirmed by T1 -weighted magnetic resonance and fluorescence imaging. The BK@AIE NPs exhibit high photothermal conversion efficiency under 980 nm NIR laser irradiation, facilitating the treatment of deep-seated tumors. Tumor progression can be effectively inhibited to extend the survival span of mice after spatiotemporal PTT. NIR irradiation can eradicate tumor tissues and release tumor-associated antigens. It is observed that the PTT treatment of GBM-bearing mice activates natural killer cells, CD3+ T cells, CD8+ T cells, and M1 macrophages in the GBM area, increasing the therapeutic efficacy. This study demonstrates that NIR-assisted BK@AIE NPs represent a promising strategy for the improved systematic elimination of GBMs and the activation of local brain immune privilege.

A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA.

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL-1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.

Self-Assembly Protein Superstructures as a Powerful Chemodynamic Therapy Nanoagent for Glioblastoma Treatment.

Glioblastoma (GBM) remains a formidable challenge in oncology. Chemodynamic therapy (CDT) that triggers tumor cell death by reactive oxygen species (ROS) could open up a new door for GBM treatment. Herein, we report a novel CDT nanoagent. Hemoglobin (Hb) and glucose oxidase (GOx) were employed as powerful CDT catalysts. Instead of encapsulating the proteins in drug delivery nanocarriers, we formulate multimeric superstructures as self-delivery entities by crosslinking techniques. Red blood cell (RBC) membranes are camouflaged on the protein superstructures to promote the delivery across blood-brain barrier. The as-prepared RBC@Hb@GOx nanoparticles (NPs) offer superior biocompatibility, simplified structure, and high accumulation at the tumor site. We successfully demonstrated that the NPs could efficiently produce toxic ROS to kill U87MG cancer cells in vitro and inhibit the growth of GBM tumor in vivo, suggesting that the new CDT nanoagent holds great promise for treating GBM.

Biodegradable Poly(γ-glutamic acid)@glucose oxidase@carbon dot nanoparticles for simultaneous multimodal imaging and synergetic cancer therapy.

It is known that tumor antigens could induce obvious anti-tumor immune responses for efficient cancer immunotherapy when combined with checkpoint blockade. However, the amount of tumor antigens is often limited due to the suppressive tumor microenvironment (TME). Here, a new type of nanomaterial was developed to improve tumor treatment by the combined action of starving therapy/photodynamic therapy (PDT)/photothermal therapy (PTT) and checkpoint-blockade immunotherapy. In detail, the immunoadjuvant nanoagents (γ-PGA@GOx@Mn,Cu-CDs) were fabricated by integrating the gamma-glutamyl transferase (GGT) enzyme-induced cellular uptake polymer-poly (γ-glutamic acid) (γ-PGA), a glucose-metabolic reaction agent - glucose oxidase (GOx), Mn,Cu-doped carbon dots (CDs) as photosensitizer and self-supplied oxygenator nanodots. γ-PGA@GOx@Mn,Cu-CDs nanoparticles (NPs) showed long retention time at the tumor acidic microenvironment and could further target cancer cells. The NPs also displayed both photothermal and photodynamic effects under laser irradiation at 730 nm. Interestingly, the endogenous generation of hydrogen peroxide (H2O2) caused by the nanoreactors could significantly relieve tumor hypoxia and further enhance in vivo PDT. By synergistically combining the NPs-based starving-like therapy/PDT/PTT and check-point-blockade therapy, the treatment efficiency was significantly improved. More importantly, the systematic antitumor immune response would eliminate non-irradiated tumors as well, which is promising for metastasis inhibition.

A multifunctional molecularly imprinted polymer-based biosensor for direct detection of doxycycline in food samples.

In this study, we developed a new type of multifunctional molecularly imprinted polymer (MIP) composite as an all-in-one biosensor for the low-cost, rapid and sensitive detection of doxycycline in pig plasma. The MIP composite consisted of a magnetic core for ease of manipulation, and a shell of fluorescent MIPs for selective recognition of doxycycline. By simply incorporating a small amount of fluorescent monomer (fluorescein-O-acrylate), the fluorescent MIP layer was successfully grafted onto the magnetic core via a surface imprinting technique. The resultant MIP composites showed significant doxycycline-dependent fluorescence quenching in an aqueous environment. Good linearity ranging from 0.2 to 6 µM was achieved, and the limit of detection was determined to be 117 nM. The biosensor also showed good selectivity towards doxycycline when compared to other common antibiotic residues. The multifunctional MIP composites were used to directly extract doxycycline from spiked pig plasma samples and quantify the antibiotics based on the quenched fluorescence signals. Recoveries of doxycycline were found in the range of 88-107%.

An immunosensor for parasite lactate dehydrogenase detection as a malaria biomarker - Comparison with commercial test kit.

This paper describes the development of an affinity sensor for the detection of Plasmodium falciparum parasite lactate dehydrogenase (pLDH) as one of the biomarkers used for malaria detection. The gold sensor was functionalised with anti-pLDH after cleaning the electrode surface to remove impurities (120 °C, 1 h). The sensor was then treated to block unreacted groups on the surface and minimise matrix interference, before applying it in a sandwich assay to detect pLDH in buffer samples using a dose concentration assay. The sensor was optimised to achieve the best detection sensitivity before using it for pLDH detection in serum samples. The developed sensor achieved a limit of detection (LOD) of 1.80 ng mL-1 and 0.70 ng mL-1 for the detection of pLDH in buffer and in serum samples respectively. The sensor sensitivity was enhanced further with the use of AuNP conjugated to the detection anti-pLDH-enzyme, achieving an LOD of 19 pg mL-1 in buffer and 23 pg mL-1 in serum samples. The performance of the sensor was compared to commercially available Plasmodium immunochromatographic (ICT) malaria kits. The developed sensor was able to detect pLDH in the Dd2luc culture medium supernatant at 0.002% parasitaemia without the use of AuNP signal enhancement when compared to the OptiMAL-IT ICT kit (detect pLDH) and the BinaxNOW ICT kit (detection of both pLDH and PfHRP 2) samples. Therefore, the sensor developed in this work is highly sensitive and can be used for pLDH detection for on-site diagnosis of malaria. A cheap and simple device as developed in this work is required to tackle malaria detection.