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Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants—containing either substitution D395N or substitutions D395N+E456N—that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications. [ABSTRACT FROM AUTHOR]

Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5′ and 3′ ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5′ end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5′ end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses. [ABSTRACT FROM AUTHOR]

Thymomas are tumors of the mediastinum often associated with autoimmune conditions, in particular myasthenia gravis. In contrast, among the fewer than 40 reports of metaplastic thymoma, myasthenia gravis is rarely found. We describe the fourth patient, and first man, with metaplastic thymoma and myasthenia gravis. A 34-year-old had acute onset of double vision with associated dysphagia and was found to have an elevation of serum acetylcholine receptor antibodies. He underwent a transsternal thymectomy. Tissue sections showed a biphasic proliferation of keratin-positive epithelial cells with a complement of spindle cells confirming the diagnosis of metaplastic thymoma. Terminal deoxynucleotidyl transferase (TDT)-positive T lymphocytes were rare and only found in the periphery of the tumor, consistent with thymic remnant. A YAP1::MAML2 gene fusion, with an in-frame fusion between genes YAP1 Exon5 (NM_001130145) and MAML2 Exon2 (NM_032427) was found, supporting further the diagnosis of metaplastic thymoma (Anchored multiplex RNA sequencing [Archer Dx, Boulder, CO] assay). The patient's gender and relatively young age, the presence of an autoimmune condition, and the lack of lymphocytic infiltrate all contribute unusual features to this case and suggest avenues for further exploration. [ABSTRACT FROM AUTHOR]

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme’s selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants—containing either substitution D395N or substitutions D395N+E456N—that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site‐specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to "smart" functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly. [ABSTRACT FROM AUTHOR]

T‐acute lymphoblastic leukemia/lymphoma (T‐ALL/LBL) is a malignant neoplasm of immature lymphoblasts. Terminal deoxynucleotidyl transferase (TDT) is a template‐independent DNA polymerase that plays an essential role in generating diversity for immunoglobulin genes. T‐ALL/LBL patients with TDT− have a worse prognosis. However, how TDT− promotes the disease progression of T‐ALL/LBL remains unknown. Here we analyzed the prognosis of T‐ALL/LBL patients in Shanghai Children's Medical Center (SCMC) and confirmed that TDT− patients had a higher rate of recurrence and remission failure and worse outcomes. Cellular experiments demonstrated that TDT was involved in DNA damage repair. TDT knockout delayed DNA repair, arrested the cell cycle and decreased apoptosis to induce the accumulation of chromosomal abnormalities and tolerance to abnormal karyotypes. Our study demonstrated that the poor outcomes in TDT− T‐ALL/LBL might be due to the drug resistance (VP16 and MTX) induced by chromosomal abnormalities. Our findings revealed novel functions and mechanisms of TDT in T‐ALL/LBL and supported that hematopoietic stem cell transplantation (HSCT) might be a better choice for these patients. [ABSTRACT FROM AUTHOR]

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

Terminal deoxynucleotidyl transferase (TdT) is a unique type of DNA polymerase predominantly expressed in precursor lymphoid cells and acute lymphoblastic leukemia. It participates in the junctional diversity of T-cell receptors and immunoglobulins. Recently, aberrant TdT expression was found in seminomas. Here, we evaluated the expression of TdT in our cohort of germ cell tumors (GCTs) with two anti-TdT antibody clones. We included 173 cases of testicular GCTs, 5 ovarian dysgerminomas, and one gonadoblastoma in the study. Tissue microarrays containing representative tumor samples were constructed and subsequently stained with anti-TdT monoclonal rabbit antibody EP266 (Dako) and TdT rabbit polyclonal antibody (Cell Marque). Expression was assessed with the H-score. No specific nuclear reaction was observed for the polyclonal anti-TdT antibody. The H-score values varied between the histological subtypes for the EP266 antibody. Positive nuclear staining was consistently seen in germ cell neoplasia in situ , seminoma, dysgerminoma, and embryonal carcinoma. Pure tumors had higher TdT H-scores than the mixed ones. Teratomas, yolk sac tumors, and choriocarcinomas were almost uniformly negative. Our study confirms that aberrant expression of TdT by testicular and ovarian GCTs exemplifies a potential diagnostic pitfall in histopathological diagnostics.

Abstract A microarray detection method based on on-chip signal amplification using terminal deoxynucleotidyl transferase (TdT) was developed to visualize pathogenic genes. Cyclic olefin copolymer (COC) substrate for microarrays was treated with oxygen plasma to induce hydrophilic surface properties. The capture probe DNA was immobilized on the COC surface by UV irradiation. The 3ʹ end of the capture probe DNA immobilized on the COC surface was modified with a phosphate group to provide resistance against the TdT reaction. Therefore, the TdT reaction was triggered only when the capture probe DNA acquired the target gene, and biotin-11-deoxyuridine triphosphate (b-dUTP) was continuously added to the 3ʹ end of the target gene. Thereafter, streptavidin-conjugated gold nanoparticles (s-AuNPs) tagged the poly uridine tails by the biotin–streptavidin interaction. The visual signal was amplified by silver enhancement in the presence of the s-AuNPs. The usefulness of this detection method was confirmed by analyzing four pathogens and allowing their visual identification.

A fluorescence aptasensor based on taking the advantage of the combination of magnetic nanoparticles (MNPs), terminal deoxynucleotidyl transferase (TdT), and CRISPR/Cas12a was developed for the determination of nasopharyngeal carcinoma (NPC)-derived exosomes. The MNPs can eliminate background interference due to their magnetic separation capability. TdT can form an ultra-long polynucleotide tail which can bind with multiple crRNA, generating a signal amplification effect. The trans-cleavage activity of CRISPR/Cas12a can be specifically triggered via the crRNA binding with DNA, resulting in the bi-labeled DNA reporter with fluorophore and quencher being cleaved. The excitation wavelength of the fluorescence spectra was 490 nm. Fluorescence spectra with emission wavelengths ranging from 511 to 600 nm were collected. Under the optimization condition, the fabricated fluorescence aptasensor for NPC-derived exosome determination exhibited excellent sensitivity and specificity, with the linear range between 500 to 5 × 104 particles mL−1 and the limit of detection of 100 particles mL−1. It can be used for the determination of NPC-derived exosomes in clinical samples, which has a considerable clinical potential and prospect. [ABSTRACT FROM AUTHOR]

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01- class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs. [ABSTRACT FROM AUTHOR]

Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia. Herein, a pho tocatalytic co lorimetry and f luorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity. Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery. • Photocatalytic colorimetry and fluorescence dual-signal biosensor was developed. • TdT-triggered polymerization reaction without DNA/RNA template. • The dsDNA/SG-I complex has excellent fluorescence-photocatalytic dual performance. • PhoCoF-sensor well integrated with centrifugal microfluidic chip for TdT detection. [ABSTRACT FROM AUTHOR]

Terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase, is recognized as a promising biomarker for acute leukemia. Herein, taking the advantage of the self-mediated strand elongation property of TdT, a simple and sensitive method for TdT activity assay was developed based on gold nanoparticles (AuNPs) labeling inductively coupled plasma mass spectrometry (ICP-MS). In the presence of TdT, the primer DNA on magnetic beads is elongated with an adenine-rich single stranded long chain that can label poly-thymine modified AuNPs. After acid elution, the labeled AuNPs were detected by ICP-MS, and the signal intensity of 197Au reflected the TdT activity. Under the optimal conditions, the limit of detection for TdT activity is down to 0.054 U mL−1, along with good selectivity and strong tolerance to other interfering proteins. Furthermore, it achieves a straightforward and accurate detection of TdT activity in acute lymphoblastic leukemia cells without sample pre-processing and tool enzyme addition. Therefore, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia therapeutics. [Display omitted] • An element labeling ICP-MS strategy is developed for TdT activity detection. • TdT activity is detected based on self-mediated strand elongation without tool enzyme. • The method is simple, sensitive and selective. • The TdT activity in acute lymphoblastic leukemia cells was determined. [ABSTRACT FROM AUTHOR]

Isolated MYC rearrangement without other recurrent genetic abnormalities is rare in B lymphoblastic leukemia/lymphoma (B-ALL/LBL), with most cases reported in pediatric patients. We report 3 adult cases with lymphoblasts showing a precursor B cell immunophenotype, and isolated MYC/IGH translocation. All 3 cases occurred in male patients with initial presentation of diffuse lymphadenopathy. Cases 1 and 2 had B-ALL with significantly increased lymphoblasts in peripheral blood and bone marrow. Case 3, a patient with human immunodeficiency virus infection, had the diagnosis of B-LBL made on a retroperitoneal lymph node biopsy and had no peripheral blood or bone marrow involvement. The leukemic and lymphoma cells in all 3 cases demonstrated Burkitt lymphoma-like morphology with deeply basophilic cytoplasm and numerous cytoplasmic vacuoles. However, all 3 had immature immunophenotypes including expression of terminal deoxynucleotidyl transferase (TdT), absence of BCL6, and dim-to-negative CD45. CD20 was largely negative in 2 of 3 cases. All 3 had confirmed MYC/IGH translocation, but lacked rearrangements of BCL2 or BCL6. EBV was negative by Epstein-Barr virus encoded small RNA in situ hybridization. Treatment protocols varied, including both high-risk ALL-type (protocol 8707) and high-grade lymphoma regimens (hyper-CVAD [cyclophosphamide, vincristine, adriamycin, and dexamethasone]), but no patient achieved continuous complete remission. These cases seem to represent a distinct biological phenomenon, in which a MYC translocation may be acquired at an immature stage of differentiation, thus manifesting features of both B-ALL/LBL and Burkitt lymphoma.

A microarray detection method based on on-chip signal amplification using terminal deoxynucleotidyl transferase (TdT) was developed to visualize pathogenic genes. Cyclic olefin copolymer (COC) substrate for microarrays was treated with oxygen plasma to induce hydrophilic surface properties. The capture probe DNA was immobilized on the COC surface by UV irradiation. The 3ʹ end of the capture probe DNA immobilized on the COC surface was modified with a phosphate group to provide resistance against the TdT reaction. Therefore, the TdT reaction was triggered only when the capture probe DNA acquired the target gene, and biotin-11-deoxyuridine triphosphate (b-dUTP) was continuously added to the 3ʹ end of the target gene. Thereafter, streptavidin-conjugated gold nanoparticles (s-AuNPs) tagged the poly uridine tails by the biotin–streptavidin interaction. The visual signal was amplified by silver enhancement in the presence of the s-AuNPs. The usefulness of this detection method was confirmed by analyzing four pathogens and allowing their visual identification. [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3′-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3′-PO 4 end of the substrate and generates 3′-OH, TdT can effectively elongate the 3′-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3′-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10−3 U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection. [Display omitted] • A highly sensitive fluorescence assay was developed for detecting ALP activity. • ALP triggered the exponential signal amplification with TdT and Endo IV. • ALP activity was detected in cell lysates containing as few as one cell. • Substrate structure preference of TdT was investigated to enhance assay sensitivity. [ABSTRACT FROM AUTHOR]

Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3' end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Enzymatic DNA writing technologies based on the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) have the potential to advance DNA information storage. TdT is unique in its ability to synthesize single-stranded DNA de novo but has limitations, including catalytic inhibition by ribonucleotide presence and slower incorporation rates compared to replicative polymerases. We anticipate that protein engineering can improve, modulate, and tailor the enzyme's properties, but there is limited information on TdT sequence-structure-function relationships to facilitate rational approaches. Therefore, we developed an easily modifiable screening assay that can measure the TdT activity in high-throughput to evaluate large TdT mutant libraries. We demonstrated the assay's capabilities by engineering TdT mutants that exhibit both improved catalytic efficiency and improved activity in the presence of an inhibitor. We screened for and identified TdT variants with greater catalytic efficiency in both selectively incorporating deoxyribonucleotides and in the presence of deoxyribonucleotide/ribonucleotide mixes. Using this information from the screening assay, we rationally engineered other TdT homologues with the same properties. The emulsion-based assay we developed is, to the best of our knowledge, the first high-throughput screening assay that can measure TdT activity quantitatively and without the need for protein purification.

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3′ blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3′OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3′ terminus of the probe fragments, and then reacted with streptavidin–phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10−3 U/ml. Moreover, it could be applied for analyzing DNA glycosylase activity in complex HeLa cell lysate samples. Therefore, this strategy possesses the advantages of high sensitivity, specificity, and multiplex, holding great potential for DNA glycosylase-related biomedical research. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are regarded as a rising star in the biomedical industry. By monitoring slight increases in miRNA-21 levels, the possibilities of multi-type malignancy can be evaluated more precisely and earlier. However, the inconvenience and insensitivity of traditional methods for detecting miRNA-21 levels remains challenging. In this study, a partially complementary cDNA probe was designed to detect miRNA-21 with target-triggered dual amplification based on strand displacement amplification (SDA) and terminal deoxynucleotidyl transferase (TdT)-assisted amplification. In this system, the presence of miRNA-21 can hybridize with template DNA to initiate SDA, generating a large number of trigger molecules. With the assistance of TdT and dGTP, the released trigger DNA with 3′-OH terminal can be elongated to a superlong poly(guanine) sequence, and a notable fluorescence signal was observed in the presence of thioflavin T. By means of dual amplification strategy, the sensing platform showed a good response tomiRNA-21 with a detection limit of 1.7 pM (S/N = 3). Moreover, the specificity of this method was verified using a set of miRNA with sequence homologous to miRNA-21. In order to further explore its practical application capabilities, the expression of miRNA in different cell lines was quantitatively analyzed and compared with the qRT-PCR. The considerable results of this study suggest great potential for the application of the proposed approach in clinical diagnosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Terminal deoxynucleotidyl transferase (TdT) is a unique type of DNA polymerase predominantly expressed in precursor lymphoid cells and acute lymphoblastic leukemia. It participates in the junctional diversity of T-cell receptors and immunoglobulins. Recently, aberrant TdT expression was found in seminomas. Here, we evaluated the expression of TdT in our cohort of germ cell tumors (GCTs) with two anti-TdT antibody clones. We included 173 cases of testicular GCTs, 5 ovarian dysgerminomas, and one gonadoblastoma in the study. Tissue microarrays containing representative tumor samples were constructed and subsequently stained with anti-TdT monoclonal rabbit antibody EP266 (Dako) and TdT rabbit polyclonal antibody (Cell Marque). Expression was assessed with the H-score. No specific nuclear reaction was observed for the polyclonal anti-TdT antibody. The H-score values varied between the histological subtypes for the EP266 antibody. Positive nuclear staining was consistently seen in germ cell neoplasia in situ, seminoma, dysgerminoma, and embryonal carcinoma. Pure tumors had higher TdT H-scores than the mixed ones. Teratomas, yolk sac tumors, and choriocarcinomas were almost uniformly negative. Our study confirms that aberrant expression of TdT by testicular and ovarian GCTs exemplifies a potential diagnostic pitfall in histopathological diagnostics. [ABSTRACT FROM AUTHOR]

Terminal deoxynucleotidyl transferase (TdT), a unique template-independent DNA polymerase, plays a crucial role in the human adaptive immune system and is considered a promising biomarker for the diagnosis of various forms of acute or chronic leukemia. The accurate and sensitive detection of trace TdT is of pivotal importance to fulfill the significant medical interest in understanding its pathological functions and diagnosing TdT-related diseases. We hereby present an in-line RNA-based microreactor direct mass spectrometry (MS) method and its application for ultrasensitive, accurate, and rapid analysis of trace TdT activity in leukemic cell samples. A specially designed RNA-based microreactor is fabricated by immobilizing short RNA sequence via covalent Au–S bond on the inner surface of a capillary pre-modified with three-dimensional porous layer (PL) and Au nanoparticles (AuNPs). Utilizing this PL@Au@RNA microreactor, the signal of target TdT is conversed into reporter molecules (adenine), which exhibit a strong MS response. This conversion process enables efficient signal amplification and enhances detection sensitivity. The outlet end of the PL@Au@RNA microreactor is deliberately crafted into a porous tip, serving as an electrospray ionization (ESI) interface to directly couple to ESI-MS in-line. This design facilitates the direct transmission of the generated signaling molecules into the MS system, eliminating the need for laborious sample treatment procedures. By implementing this RNA-based microreactor in direct MS analysis, we have achieved remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10−9 U, surpassing other reported methods in literature by three to four orders of magnitude. Furthermore, each assay requires a minimal sample volume of merely 10 nL. This method has successfully demonstrated its application in accurately and efficiently detecting TdT activity in leukemia cells, and its detection results are consistent with those obtained by ELISA kits. [Display omitted] • An in-line RNA-based microreactor direct MS method for ultrasensitive and rapid assay of TdT activity. • The microreactor is fabricated by immobilizing RNA on a capillary surface pre-modified with 3D porous layer and AuNPs. • TdT-adenine signal conversion and amplification in microreactor is achieved by TdT-induced polymerization and depurination. • The remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10−9 U is achieved. • Each assay requires a sample volume of merely 10 nL and eliminates the need for laborious sample treatment procedures. [ABSTRACT FROM AUTHOR]

Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge. In this study, we introduce a Terminal deoxynucleotidyl Transferase (TdT)-based amplification loop, creating a positive feedback mechanism within the CRISPR-Cas12a pathogen detection system. Upon recognizing pathogenic target DNA, Cas12a triggers trans-cleavage of a FRET reporter and a specific enhancer molecule oligonucleotide, indicated by the acronym POISER (Partial Or Incomplete Sites for crRNA recognition). POISER comprises half of a CRISPR-RNA recognition site, which is subsequently elongated by TdT enzymatic activity. This process, involving pathogen recognition-induced Cas12a cleavage and TdT elongation, results in a novel single-stranded DNA target. This target can subsequently be recognized by a POISER-specific crRNA, activating more Cas12a enzymes. Our study demonstrates that these POISER-cycles enhance the signal strength in fluorescent-based CRISPR-Cas12a assays. Although further refinement is desirable, POISER holds promise as a valuable tool for the detection of pathogens in point-of-care testing, surveillance, and environmental monitoring. • This study introduces a Terminal deoxynucleotidyl Transferasebased amplification loop within a CRISPR-Cas12a DNA detection system. By creating a positive feedback mechanism, this innovative approach enhances the signal strength in fluorescent-based CRISPR-Cas12a assays without the need for DNA pre-amplification. • The POISER system employs an enhancer oligonucleotide containing half of a crRNA recognition site. Combined with TdT activity, this system generates a novel single-stranded DNA target that enhances the signal intensity in CRISPR-Cas12a FRET assays. [added extra bullet point] By leveraging the POISER system, this study demonstrates increased signals in DNA detection assays, facilitating the development of more efficient diagnostic tools. [ABSTRACT FROM AUTHOR]

The market for using and storing digital data is growing, with DNA synthesis emerging as an efficient way to store massive amounts of data. Storing information in DNA mainly consists of two steps: data writing and reading. The writing step requires encoding data in DNA, building one nucleotide at a time as a form of single-stranded DNA (ssDNA). Once the data needs to be read, the target DNA is selectively retrieved and sequenced, which will also be in the form of an ssDNA. Recently, enzyme-based DNA synthesis is emerging as a new method to be a breakthrough on behalf of decades-old chemical synthesis. A few enzymatic methods have been presented for data memory, including the use of terminal deoxynucleotidyl transferase. Besides, enzyme-based amplification or denaturation of the target strand into ssDNA provides selective access to the desired dataset. In this review, we summarize diverse enzymatic methods for either synthesizing ssDNA or retrieving the data-containing DNA.

Alkaline phosphatase (ALP) is a zinc-containing metalloprotein that shows very great significance in clinical diagnosis, which can catalyze the hydrolysis of phosphorylated species. ALP has the potential to serve as a valuable biomarker for detecting liver dysfunction and bone diseases. On the other hand, ALP is an efficient biocatalyst to amplify detection signals in the enzyme-linked assay. It has always been a major research focus to develop novel biosensors that can detect ALP activity with high selectivity and sensitivity. There have been numerous reports on the development of biosensors to determine ALP activity using a phosphorylated DNA probe. Among them, various beneficial strategies, such as λ exonuclease-mediated cleavage reaction, terminal deoxynucleotidyl transferase-triggered DNA polymerization, and Klenow fragment polymerase-catalyzed elongation, are employed to generate amplified and more intuitive signal. This review discusses and summarizes the development and advances of biosensors for ALP activity detection that use a well-designed phosphorylated DNA probe, aiming to provide some guidelines for the design of more sophisticated sensing strategies that exhibit improved sensitivity, selectivity, and adaptability in detecting ALP activity. Taking full advantages of efficient alkaline phosphatase-catalyzed dephosphorylation, specific DNA sensing, as well as various signal amplification strategies (e.g., λ exonuclease-mediated cleavage reaction, terminal deoxynucleotidyl transferase-triggered DNA polymerization, Klenow fragment polymerase-catalyzed elongation), biosensors for ALP activity detection based on a phosphorylated DNA probe are reviewed to summarize and comment on their development and advances. [Display omitted] • Biosensors for ALP activity detection that based on a phosphorylated DNA probe have been reviewed. • The λ exo-, TdT-, and KF polymerase-based sensing strategies are discussed in detail. • The detection performance and working principle of these DNA-based biosensors are summarized. • Further prospects of phosphorylated DNA-based biosensors for ALP activity detection are presented. [ABSTRACT FROM AUTHOR]

In the modern era, classification of neoplasms not only depends on immunomorphological features but also on specific disease-defining genetic events. Translocations/rearrangements of MYC/8q24 locus combined with BCL-2 or BCL6 translocations (double/triple hit) are considered hallmarks of high-grade B-cell lymphoma (HGBL), a type of aggressive mature B-cell lymphoma. When cases with immature immunophenotypes present these rearrangements, diagnosis becomes very difficult. We herein report an unusual case of an aggressive B-cell lymphoma/leukemia that presented with immature morphology and immunophenotype with triple hit gene rearrangements. This case highlights the difficulty in classifying and appropriately treating these patients. The novel aspect is the treatment and outcome with chimeric antigen receptor or CAR T-cell therapy. [ABSTRACT FROM AUTHOR]

Purpose: T-Lymphoblastic lymphoma (T-LBL) is a rare malignant tumor originated from precursor T-lymphocytes that differentiate to T lymphocytes. We report a rare case of T-LBL presenting with conjunctival mass as the first sign. Observations: A 61-year-old man presented with a right salmon colored conjunctival mass. A biopsy was performed, and histopathologic examination showed dense lymphocyte proliferation in subepithelial substantia propria. Immunohistochemical staining was positive for CD7, CD10, and TdT; and negative for CD20. CD3 was negative in most parts PET-CT revealed abnormal uptake in the left cervix, anterior mediastinum, abdominal aortic lymph nodes, and multiple bones. From the above findings, stage IVA T-LBL was diagnosed. The patient received hyper CVAD therapy (cyclophosphamide + doxorubicin + vincristine + dexamethasone) and HD-MA therapy (high-dose methotrexate + cytarabine). Subsequently, an unrelated bone marrow transplant was performed. Conclusions and importance: This case demonstrates the importance of considering rare lymphomas such as T-LBL in the differential diagnosis of ocular adnexal lymphoid neoplasms.

In the modern era, classification of neoplasms not only depends on immunomorphological features but also on specific disease defining genetic events. Translocations/rearrangements of MYC/8q24 locus in combination with BCL-2 or BCL6 translocations (double/triple hit) are considered hallmarks of high-grade B-cell lymphoma (HGBL), a type of aggressive mature B-cell lymphoma. When cases with immature immunophenotype present with these rearrangements, establishment of diagnosis becomes very difficult. We herein report an unusual case of B-cell lymphoma that presented with immature morphology and immunophenotype with triple hit gene rearrangements. This case highlights the difficulty in classifying and appropriately treating these patients. The novel aspect of this case is the treatment and outcome with chimeric antigen receptor, or CAR T-cell therapy. Keywords: high-grade Lymphoma, TDT, MYC, BCL6.

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC − R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC − R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC − R+, n = 15, MYC − R−, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC − R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC − R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia. [ABSTRACT FROM AUTHOR]

A method is presented that uses photoinduced electron transfer (PET) for the determination of microRNAs (miRNAs) in clinical serum samples and complicated cell samples by using a smartphone. miRNA-21 is adopted as a model analyte. A 3′-phosphorylated DNA probe containing AgNCs is synthesized and hybridized with miRNA-21. Subsequently, the probe is cleaved specifically by duplex-specific nuclease to form 3′-hydroxylated products, then extended by terminal deoxynucleotidyl transferase (TdT) with superlong G for G-quadruplex/hemin units fabrication. In this way, PET occurred between AgNCs and produced G-quadruplex/hemin units, leading to the fluorescence quenching of AgNCs. Notably, the fluorescence images can be captured and translated into digital information by smartphone, resulting in a direct quantitative determination of miRNA. As a result, our strategy for miRNA assay is achieved with a satisfactory detection limit of 1.43 pM. Interestingly, TdT-propelled G-quadruplex/hemin units as multiple electron acceptors promote the sensitivity of miRNA monitoring. Different miRNAs assays are realized by adjusting the complimentary sequences of DNA probe. These qualities not only broaden the practical application of PET-based strategy, but also provide a new insight into the nucleic acid detection. [ABSTRACT FROM AUTHOR]

Excessive lead ions (Pb2+) would make the human body suffer from anemia and neurological disorders. Therefore, it was of great significance to study an efficient method for the point-of-care (POC) detection of Pb2+. In this paper, an innovative method for sensitive detection of Pb2+ in river water was proposed, which combined blood glucose meter signal transduction with terminal deoxynucleotidyl transferase (TdT) mediated DNA extension amplification strategy. The gold nanoparticle-modified electrode was used as the sensing interface, which was fabricated through the choline monolayer electrodeposition method. The target Pb2+ initiated DNAzyme cyclic cleavage reaction on the magnetic bead, causing the substrate probe to generate 3′-OH terminus, which would induce the formation of polyadenine (polyA) sequences with the assistance of TdT. Ultimately, the numerous invertase tags on magnetic beads would be loaded on the sensing interface through the interaction between polyA and gold nanoparticles, which would catalyze the hydrolysis of sucrose to glucose with highly efficient enzymatic turnover, enabling the blood glucose meter for detection of Pb2+. The method exhibited a detection limit down to 0.6 pM, and can be used to detect Pb2+ in river water and tap water with high selectivity, offering the method great potential to be applied in environmental monitoring and point-of-care diagnostic applications. • A point-of-care (POC) testing sensor for Pb2+ detection was constructed. • This sensor displayed a low detection limit of 0.6 pM with the advantages of portability, convenience, and low cost. • The excellent selectivity and anti-interference ability achieved the detection of Pb2+ in tap water and river water. • The gold nanoparticle-modified electrode was fabricated through the choline monolayer electrodeposition method. • The sensor was promising for application in environmental monitoring and point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans -cleavage of a reporter molecule. By combining these processes, the target genomic DNA extracted from V. parahaemolyticus is able to activate many CRISPR/Cas12a units (CRISPR/Cas12an) simultaneously, resulting in a greatly amplified target signal to indicate the presence and concentration of V. parahaemolyticus. This unique model offers more advantages compared to traditional amplification models that use one RPA amplicon to activate one CRISPR/Cas12a unit. Under optimized conditions, our method enables the detection of target V. parahaemolyticus within a linear range of 1 × 102–1 × 107 CFU/mL, with an impressive limit of detection as low as 12.4 CFU/mL. It is conceivable that the adaptive tandem CRISPR/Cas12a molecular amplifier could be adapted as routine diagnostic kits in future for in-field detection of pathogens. [Display omitted] • An adaptive tandem CRISPR/Cas12a molecular amplifier has been designed. • The molecular amplifier involves the co-activation of multiple CRISPR/Cas12a units to trans -cleavage of a reporter molecule. • The molecular amplifier is advantageous than traditional one dsDNA amplicon to activate one CRISPR/Cas12a unit. • The method enables the detection of Vibrio parahaemolyticus with a robust assay ability. [ABSTRACT FROM AUTHOR]

Background/Aims: T-cell acute lymphoblastic leukemia (T-ALL) is an uncommon, aggressive malignancy that rarely presents in the skin and is generally not considered as part of the differential diagnosis by dermatologists and dermatopathologists. We describe an unusual case of T-ALL presenting with folliculocentric, erythematous papules on the face, histologically resembling mycosis fungoides (MF). Immunostaining for terminal deoxynucleotidyl transferase (TdT) was positive in tumor cells, supporting the diagnosis of cutaneous involvement by T-ALL. TdT is a nuclear enzyme expressed by immature lymphoid malignancies, but the expression pattern of this marker is not well characterized in the skin. We aimed to assess TdT staining in skin biopsies with similar-appearing lymphocytic infiltrates. Methods: We evaluated the immunostaining profile of TdT in a cohort of 23 patients, including 13 cases of MF and 10 cases of spongiotic dermatitis. Results: The lymphocytes in the MF and spongiotic dermatitis cases lacked nuclear staining for TdT. Nonspecific, granular, cytoplasmic staining was observed in a small number of background cells. Conclusions: TdT may assist dermatopathologists in discriminating malignant infiltrates of T-ALL from other conditions.

MicroRNAs (miRNAs) are regarded as a rising star in the biomedical industry. By monitoring slight increases in miRNA-21 levels, the possibilities of multi-type malignancy can be evaluated more precisely and earlier. However, the inconvenience and insensitivity of traditional methods for detecting miRNA-21 levels remains challenging. In this study, a partially complementary cDNA probe was designed to detect miRNA-21 with target-triggered dual amplification based on strand displacement amplification (SDA) and terminal deoxynucleotidyl transferase (TdT)-assisted amplification. In this system, the presence of miRNA-21 can hybridize with template DNA to initiate SDA, generating a large number of trigger molecules. With the assistance of TdT and dGTP, the released trigger DNA with 3′-OH terminal can be elongated to a superlong poly(guanine) sequence, and a notable fluorescence signal was observed in the presence of thioflavin T. By means of dual amplification strategy, the sensing platform showed a good response tomiRNA-21 with a detection limit of 1.7 pM (S/N = 3). Moreover, the specificity of this method was verified using a set of miRNA with sequence homologous to miRNA-21. In order to further explore its practical application capabilities, the expression of miRNA in different cell lines was quantitatively analyzed and compared with the qRT-PCR. The considerable results of this study suggest great potential for the application of the proposed approach in clinical diagnosis.

Terminal deoxynucleotidyl transferase (TdT) is expressed on precursor lymphoblastic neoplasms and some acute myeloid leukemia (AML) cells. The clinical impact of TdT expression on AML outcomes remains unclear. Here, we conducted a retrospective analysis to identify prognostic implications of TdT expression in AML with an intermediate-risk karyotype. Forty-eight cases of intermediate-risk AML were enrolled. TdT positivity was defined as expression on ≥ 10% of the gated cells. Of 48 cases, 12 (25%) were positive for TdT [median expression rate of TdT 0.9% (range 0-86.9%)]. No significant differences in patient characteristics or complete remission rate were observed between TdT-positive and TdT-negative cases. The probability of overall survival (OS) and event-free survival (EFS) at 1 year was not significantly different between TdT-positive and TdT-negative cases (OS: 58.3% vs. 65.2%, p = 0.32; EFS: 33.3% vs. 57.1%, p = 0.06). Relapse-free survival (RFS) probability at 1 year was significantly lower for TdT-positive than TdT-negative cases (10% vs. 71.3%, p = 0.002). Multivariate analyses revealed that TdT positivity was an independent significant adverse factor for RFS [hazard ratio: 3.309, 95% confidence interval: 1.334-8.209, p = 0.009]. Our results suggest that TdT expression is associated with increased risk of relapse in patients with intermediate-risk AML. [ABSTRACT FROM AUTHOR]

A novel indirect electrochemical approach is reported to improve the sensitivity of Hg2+ quantification in environmental water samples. This strategy is based on the Hg2+ induced selective interaction of thymine mismatched pairs in a thymine-rich aptamer DNA to improve the specificity, while template-independent terminal deoxynucleotidyl transferase (TdT) extended the 3′-OH aptamer termini with repeated bases and increased the sensitivity. The introduction of the target Hg2+ blocked aptamer extension. Taking advantage of an electrochemical platform, this aptamer-TdT based amplification sensor enabled the specific quantification of Hg2+ ion over a wide linear range from 2 pM to 20 nM with a low limit of detection equal to 0.1 pM. The proposed strategy is promising for the detection of other metal ions. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world, which can lead to considerably high mortality rate. It was reported that the prognosis is extremely poor and survival is often measured in months once CRC metastases become clinically evident. Therefore, the development of effective approach for metastatic CRC cells detection and imaging may potentially be significant and helpful for CRC prognosis and treatment. Therefore, we proposed a sensitive and specific approach for high-metastatic CRC LoVo cells detection and imaging by using terminal deoxynucleotidyl transferase (TdT)-initiated molecule beacons (MBs) arrayed fluorescent aptamer probes (denoted as TMAP). In this approach, the aptamer W3 targeting high-metastatic CRC LoVo cells was elongated to form W3-poly A at the 3′-hydroxyl terminus with repeated A bases in the presence of TdT and dATP. The MBs designed with poly T sequence in the loop were then hybridized with the poly A in the aptamer W3. The TMAP was easily constructed without the need of aptamer modification. It was demonstrated that this approach could specifically detect and image the high-metastatic CRC LoVo cells from the mixture of high-metastatic CRC LoVo cells and non-metastatic HCT-8 cells. Compared with 6-carboxyfluorescein (6-FAM) labeled aptamer W3, the TMAP was demonstrated to have a much stronger fluorescence signal on the target cells, realizing a 4-fold increase in signal-to-background ratio (SBR). Determination by flow cytometry allowed for detection of as low as 23 CRC LoVo cells in 200 μL cell culture medium. The high sensitivity and the capability for using in complicate biological samples imply that this approach holds considerable potential for metastatic CRC detection and therapy. Image 1 • TMAP was developed for specific detection and imaging of metastatic CRC cells. • TMAP could identify target LoVo cells with excellent binding affinity and specificity. • TMAP could specifically distinguish between the metastatic LoVo cells and non-metastatic HCT-8 cells in mixed cells samples. [ABSTRACT FROM AUTHOR]

Prostate-specific antigen (PSA) is the only biomarker for the diagnosis of prostate cancer. So the PSA screening test is very important due to the high occurrence of prostate cancer in men. In this work, a label-free fluorescent method was developed based on terminal deoxynucleotidyl transferase (TdT) and G–quadruplex–thioflavin T complex for detecting PSA. In the absence of PSA, the PSA aptamer can be used as the primer for TdT extension reactions, resulting in the formation of G-quadruplexes and generation of strong fluorescent signals. After the addition of PSA, the PSA–aptamer complex prevented the TdT extension reaction due to steric hindrance, thus resulting in a poor fluorescent signal. The assay showed a wide linear range (0.1 to 80 pg/mL) and a detection limit of 0.086 pg/mL (S/N = 3). It also has good specificity for PSA determination and gives satisfactory results when applied to biological samples. Conceivably, its merits such as good selectivity and high sensitivity indicate that the proposed method has a promising application potential in the clinical diagnosis and treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

• A label-free and highly sensitive fluorescence strategy was developed for detection of Dam MTase activity and screen of its inhibitors. • The excellent amplified efficiency was achieved owing to the dual powerful signal enhancement of RCA and G-triplex@ThT. • The template-independent property of TdT and easy preparation of dumbbell-like RCA template facilitate the simple design of the strategy. • The proposed strategy displayed potential applicability of detecting target Dam MTase in complex biological matrix. It is of great significance to sensitively detect DNA methyltransferase (MTase) activity and screen its inhibitory drugs in the field of human cancer diagnosis and disease therapeutics. In this work, based on terminal deoxynucleotidyl transferase (TdT)-induced rolling circle amplification (RCA), we construct a label-free and highly sensitive fluorescence biosensing strategy for DNA MTase activity analysis. The hairpin substrate is methylated by Dam MTase and cleaved by Dpnl to generate a DNA fragment with a free 3′-hydroxy (3′−OH) terminus that is extended by TdT to obtain a long adenine (poly-A) tail. Subsequently, the resulting poly-A work as primer triggers RCA reaction and generate lots of G-triplexes which can combine with Thioflavin T (ThT) to achieve the apparent fluorescence signal. Integrate TdT-induced RCA with G-triplex@ThT, Dam MTase can be detected down to 0.058 U/mL with a wide linear range from 0.1 U/mL to 40 U/mL. In addition, the method exhibits high selectivity to other MTases. We also successfully demonstrate this work can be used in complex matrixes assay and screen MTase inhibitors. Thus, in the area of DNA MTase-related cancer diagnosis and disease therapeutics, the developed method may be a promising tool for the detection of Dam MTase. [ABSTRACT FROM AUTHOR]

In this work, a dynamic terminus-regulated fabric of DNA hydrogel was invented in debt to the reiterative catalysis of terminal deoxynucleotidyl transferase (TdT). It extended free 3'−OH end to an overhang of homopolymeric adenosine base pair, and alternated with branching from the frayed complementary seed oligo T 20 G 5. The cycle of this template-independent and isothermal amplification resulted in a microscale dendritic DNA fractal at first, which then gelatinized into a cohesive and intricate 3D network. Details of the complex were elucidated with gel electrophoresis, confocal and atomic force microscopy. Its well hydrated inner space could further provide plenty of biocompatible chambers for enzymatic transducers fused along the elongation. Taking merits of this neat and flexible setup, an in situ hydrogelation strategy was developed and utilized in the signal cascade of a miRNA biomarker detector on an electrode microarray, thus accomplished an ultrasensitive, selective and high-throughput sensing even for real samples. This collective manipulation of DNA-protein hydrogel ensemble on interface demonstrates its potency as a general scheme of sensitization in bioanalytical applications. • A dynamic terminus-regulated fabric of DNA hydrogel was designed. • An in situ hydrogelation strategy was used in the signal cascade of a miRNA sensor. • It facilitated the fabrication of an ultrasensitive, selective and high-throughput detection. [ABSTRACT FROM AUTHOR]

Background/Aims: T-cell acute lymphoblastic leukemia (T-ALL) is an uncommon, aggressive malignancy that rarely presents in the skin and is generally not considered as part of the differential diagnosis by dermatologists and dermatopathologists. We describe an unusual case of T-ALL presenting with folliculocentric, erythematous papules on the face, histologically resembling mycosis fungoides (MF). Immunostaining for terminal deoxynucleotidyl transferase (TdT) was positive in tumor cells, supporting the diagnosis of cutaneous involvement by T-ALL. TdT is a nuclear enzyme expressed by immature lymphoid malignancies, but the expression pattern of this marker is not well characterized in the skin. We aimed to assess TdT staining in skin biopsies with similar-appearing lymphocytic infiltrates. Methods: We evaluated the immunostaining profile of TdT in a cohort of 23 patients, including 13 cases of MF and 10 cases of spongiotic dermatitis. Results: The lymphocytes in the MF and spongiotic dermatitis cases lacked nuclear staining for TdT. Nonspecific, granular, cytoplasmic staining was observed in a small number of background cells. Conclusions: TdT may assist dermatopathologists in discriminating malignant infiltrates of T-ALL from other conditions. [ABSTRACT FROM AUTHOR]

Abstract Alkaline phosphatase (ALP) is a vital hydrolysis enzyme in phosphate metabolism, which catalyzes the hydrolysis of phosphate ester groups in proteins, nucleic acids, and other small molecules. Meanwhile, abnormal ALP expression is associated with occurrence and development of many diseases. Terminal deoxynucleotidyl transferase (TdT) is a widely used tool enzyme in many fields, which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3′-OH termini of ssDNA in a template-free manner. In this work, we designed a versatile, convenient, label-free, and highly sensitive fluorescence enhancing assay for ALP activity detection based on the characteristics of ALP, TdT, and thymine-HgII-thymine (T-Hg2+-T) structure. In the presence of ALP, the 3′-phosphoryl end of the ssDNA-p was hydrolyzed to hydroxyl group, followed by addition of a poly-T tail on its 3′ terminal hydroxyl in the mixing solution containing both TdT and dTTPs. Then, the DNA with poly-T tail could interact with Hg2+ to form the stable T-Hg2+-T mediated metallo DNA duplex, which enhanced the fluorescence intensity of the SG. Under optimal conditions, the proposed system was employed for quantitatively monitoring ALP activity with a dynamic range of 0–2500 mU mL−1, and the actual detection limit could be down to 0.025 mU mL−1. And the determination of ALP activity in human serum samples and MCF-7 cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis. Meanwhile, this system could also be applied to both TdT and Hg2+ detection. Graphical abstract fx1 Highlights • ALP was sensitive detected in this assay and as low as 0.025 mU mL−1 of ALP could easily detected on fluorescent spectrometer. • TdT and Hg2+could also be detected synchronously within the same system. • ALP in serum and organism samples could be evaluated either. [ABSTRACT FROM AUTHOR]

Abstract Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3′-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis. Graphical abstract Image 1 Highlights • Developing a new label-free, facile, rapid TdT detection assay. • KSCNis a better coralyne quenching agent. • This assay could be directly used in cell lysates sample's detection. [ABSTRACT FROM AUTHOR]

A label‐free approach that takes advantage of intrinsic electrochemical activity of nucleobases has been applied to study the products of terminal deoxynucleotidyl transferase (TdT) tailing reaction. DNA homooligonucleotides A30, C30 and T30 were used as primers for the tailing reaction to which a dNTP – or a mixture of dNTPs – and TdT were added to form the tails. Electrochemical detection enabled study of the tailing reaction products created by various combinations of primers and dNTPs, with pyrolytic graphite electrode (PGE) being suitable for remarkably precise analysis of the length of tailing reaction products. Furthermore, the hanging mercury drop electrode (HMDE) was able to reveal formation of various DNA structures, such as DNA hairpins and G‐quadruplexes, which influence the behavior of DNA molecules at the negatively charged surface of HMDE. Thus, the described approach proves to be an excellent tool for studying the TdT tailing reactions and for exploring how various DNA structures affect both the tailing reactions and electrochemical behavior of DNA oligonucleotides at electrode surfaces. [ABSTRACT FROM AUTHOR]

Abstract This study describes a novel electrochemical aptasensor for detection of α-synuclein (α-syn) oligomer, an important biomarker related to Parkinson's and Alzheimer's diseases. The sensing platform is based on exonuclease I (Exo I), terminal deoxynucleotidyl transferase (TdT) and methylene blue. The aptasensor exploits the improved sensitivity because of applications of TdT and Exo I and also a label-free aptamer (Apt). Furthermore, direct immobilization of complementary strand of aptamer (CS) instead of Apt on the surface of electrode prohibits Apt self-assembled monolayer aggregation and keeps the function of the Apt. In the absence of α-syn oligomer, TdT enhances lengths of Apt and CS and so, increases accumulation of methylene blue as redox agent on the surface of electrode, leading to a strong current signal. While in the presence of α-syn oligomer, Exo I digests CS on the electrode surface, resulting in less accumulation of methylene blue on the electrode surface and a weak current signal. The relative electrochemical signal of the aptasensor increased linearly with the logarithm of α-syn oligomer concentration in the range from 60 pM to 150 nM. The detection limit was 10 pM. Furthermore, the sensor showed high precision and repeatability for detection of α-syn oligomer in serum samples. Highlights • This study describes a novel electrochemical aptasensor for detection of α-synuclein (α-syn) oligomer. • The sensing platform is based on exonuclease I (Exo I), terminal deoxynucleotidyl transferase (TdT) and methylene blue. • The aptasensor exploits the improved sensitivity because of applications of TdT and Exo I and also a label-free aptamer. • The sensor showed high precision and repeatability for detection of α-syn oligomer in serum samples. [ABSTRACT FROM AUTHOR]