Enhancing the expression of terminal deoxynucleotidyl transferases by N-terminal truncation.

Terminal deoxynucleotidyl transferase (TdT), a unique DNA polymerase that catalyzes the template-free incorporation of nucleotides into single-stranded DNA, has facilitated the development of various oligonucleotide-based tools and methods, especially in the field of template-free enzymatic DNA synthesis. However, expressing vertebrate-derived TdTs in Escherichia coli complicates purification and increases production costs. In this study, N-terminal truncation of TdTs was performed to improve their expression and stability. The results revealed that N-terminal truncation could enhance the expression level of six TdTs. Among the truncated mutants, N-140-ZaTdT and N-140-CpTdT, with 140 amino acids removed, exhibited an increase in protein expression, which was 9.5- and 23-fold higher than their wild-types, respectively. Importantly, the truncation preserves the catalytic function of TdT. Additionally, the Tm values of N-140-ZaTdT increased by 4.9°C. The improved expression of the truncated mutants makes them more suitable for reducing production costs and advancing enzyme engineering.

Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase.

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.

In-line RNA-based microreactor direct mass spectrometry for ultrasensitive and rapid assay of terminal deoxynucleotidyl transferase activity.

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.

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

Engineering the Activity of a Template-Independent DNA Polymerase.

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.

A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels.

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.

Semiarbitrary qPCR for Sensitive Detection of Circulating miRNA via Terminal Deoxynucleotidyl Transferase-Assisted RNA-Primed DNA Polymerization.

Circulating microRNAs (miRNAs) have recently emerged as noninvasive disease biomarkers. Quantitative detection of circulating miRNAs could offer significant information for clinical diagnosis due to its significance in the development of biological processes. In response to the current challenges of circulating miRNA detection, we introduce a sensitive, selective, and versatile circulating miRNA detection strategy using terminal deoxynucleotidyl transferase (TdT)-catalyzed RNA-primed DNA polymerization (TCRDP) coupled with semiarbitrary qPCR (SAPCR). Semiarbitrary qPCR was first developed here to detect long fragment targets with only a short-known sequence or to detect a short fragment target after extension with terminal transferase. Besides, the subsequent results show that TdT has a preference for RNA, particularly for extending RNAs with purine-rich and unstructured ends. Consequently, utilizing this assay, we have successfully applied it to the quantitative analysis of circulating miR-122 in animal models, a sensitive and informative biomarker for drug-induced liver injury, and as low as 200 zmol of the target is detected with desirable specificity and sensitivity, indicating that the TCRDP-SAPCR can offer a promising platform for nucleic acids analysis.

One-pot isothermal amplification permits recycled activation of CRISPR/Cas12a for sensing terminal deoxynucleotidyl transferase activity.

This study introduces a one-pot isothermal amplification assay for ultrasensitive analysis of terminal deoxynucleotidyl transferase (TdT) activity. The system realizes recycled activation of CRISPR/Cas12a, enabling exceptional signal amplification. This approach maximizes the simplicity of the detection method, offering a promising avenue for molecular disease diagnosis.

Terminal deoxynucleotidyl transferase expression in different subtypes of childhood B-cell acute lymphoblastic leukemia.

The lack of expression of terminal deoxynucleotidyl transferase (TdT) is frequently associated with KMT2A-rearranged subtype of pediatric acute lymphoblastic leukemia (ALL). However, this association has not been investigated extensively in the Asian population. A retrospective analysis of TdT expression in pediatric B-cell ALL (B-ALL) was performed in patients treated using the Taiwan Pediatric Oncology Group (TPOG) ALL 2002 and 2013 protocols. Among the 331 patients with B-ALL, 12 patients showed TdT negativity at initial diagnosis. Among these, eight patients showed KMT2A rearrangement (66.7%). Other patients showing negative TdT expression had ETV6::RUNX1, MEF2D-rearranged, and other B-ALL subtypes. However, in the context of KMT2A-rearranged B-ALL (n = 20), only eight patients showed TdT negativity. The 5-year event-free survival and overall survival of patients with and without TdT expression were 83.8% versus 46.8% (P <0.001) and 86.3% versus 55.4% (P = 0.004), respectively. Moreover, several aberrant markers, such as CD2, CD56, CD7, and CD117, were rarely expressed in the B-ALL samples, and if expressed, they were enriched in specific genetic subtypes. The results of this study indicate that immunophenotypic features are correlated with specific genetic subtypes of childhood B-ALL.

Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its Applications.

Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.

Detection of terminal deoxynucleotidyl transferase activity based on self-mediated nucleic acid elongation and elemental labeling inductively coupled plasma-mass spectrometry.

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.

Enzymatic Synthesis of TNA Protects DNA Nanostructures.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3' terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

Substrate Specificity Diversity of Human Terminal Deoxynucleotidyltransferase May Be a Naturally Programmed Feature Facilitating Its Biological Function.

Terminal 2'-deoxynucleotidyl transferase (TdT) is a unique enzyme capable of catalysing template-independent elongation of DNA 3' ends during V(D)J recombination. The mechanism controlling the enzyme's substrate specificity, which is necessary for its biological function, remains unknown. Accordingly, in this work, kinetic and mutational analyses of human TdT were performed and allowed to determine quantitative characteristics of individual stages of the enzyme-substrate interaction, which overall may ensure the enzyme's operation either in the distributive or processive mode of primer extension. It was found that conformational dynamics of TdT play an important role in the formation of the catalytic complex. Meanwhile, the nature of the nitrogenous base significantly affected both the dNTP-binding and catalytic-reaction efficiency. The results indicated that neutralisation of the charge and an increase in the internal volume of the active site caused a substantial increase in the activity of the enzyme and induced a transition to the processive mode in the presence of Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ also may regulate the switching of the enzymatic process to the processive mode. Thus, the totality of individual factors affecting the activity of TdT ensures effective execution of its biological function.

3D-DNA walking nanomachine based on catalytic hairpin assembly and copper nanoclusters for sensitive detection of hepatitis C virus.

Rapid and accurate detection of the hepatitis C virus (HCV) is essential for early diagnosis and prevention of virus transmission. This study presents a novel approach that combines the three-dimensional (3D)-DNA walking nanomachine with catalytic hairpin assembly (CHA) and copper nanoclusters (CuNCs). By integrating CHA with the 3D DNA walking nanomachine, efficient target amplification on 3D surfaces was achieved, leading to improved reaction speed and detection performance. Terminal deoxynucleotidyl transferase (TdT) was utilized to generate T-rich DNA sequences. These sequences served as templates for the formation of CuNCs, which functioned as the readout signal. The optimized 3D-DNA walking nanomachine exhibited excellent sensitivity in detecting HCV, with a detection limit of 42.4 pM and a linear range of 100 pM to 2 nM. The biosensor demonstrated excellent selectivity and reproducibility, with a recovery rate ranging from 94% to 108% for the detection of real samples. This design holds great potential for sensitive, label-free, and reliable detection of HCV in clinical settings. Furthermore, the versatility of this approach allows for the customization of target sequences, thereby facilitating the detection of various nucleic acid targets. Therefore, this method has the potential to advance personalized medicine, disease management, and genetic analysis in the field of molecular diagnosis.

Increased Incidence of TdT-negative Pre-B Acute Lymphoblastic Leukemia Associated With Poor Prognostic Features Among Mexican Children in Central Mexico.

Mexican and Hispanic children in Mexico and the United States, respectively, have the highest incidence and worst outcomes of pre-B acute lymphoblastic leukemia (ALL) compared with other racial/ethnic groups. Terminal deoxynucleotidyl transferase (TdT) is an intranuclear DNA polymerase normally present on immature lymphocytes (TdT-positive) and distinguishes ALL from mature lymphoid malignancies. We performed a multisite retrospective study to determine the incidence of TdT-negative precursor B-cell acute lymphoblastic leukemia (pre-B ALL) among Mexican, Caucasian, and US-born Hispanic children to correlate TdT expression with patient characteristics and known prognostic factors. Fisher exact test was performed for categorical variables and the Wilcoxon rank-sum test was used for continuous variables. TdT-negative pre-B ALL was most frequently identified in patients with National Cancer Institute high-risk disease ( P =0.014). TdT-negative expression was also most frequently associated with hypodiploid pre-B ALL ( P =0.001) and KMT2A gene rearrangement ( P =0.0012). Mexican children had the highest incidence of TdT-negative ALL compared with Caucasians and US Hispanics ( P <0.001), with an increased incidence of poor prognostic features as well. This study demonstrates significant differences in TdT-negative expression, genomic alterations, and leukemic ploidy based on race and ethnicity.

Dual Self-Amplification Homogeneous Electrochemiluminescence Biosensor for Terminal Deoxynucleotidyl Transferase Activity Based on Controlling the Surface Morphology and Charge of Reporter Nanoparticles.

Terminal deoxynucleotidyl transferase (TdT) is upregulated in several types of leukemia and is considered a disease biomarker and a potential therapeutic target for leukemia. In this research, a homogeneous electrochemiluminescence (ECL) method based on the control of surface charge and morphology of tris (2,2'-bipyridine) ruthenium(II) chloride hexahydrate-doped silica nanoparticles (Ru@SiO2 NPs) has been designed for TdT activity detection. A small amount of short single-stranded DNA (ssDNA) was modified onto the surface of Ru@SiO2 NPs, and the nanoparticles with a slight positive charge experienced electrostatic attraction with the indium tin oxide (ITO) electrode with a negative charge, so relatively high ECL signals had been detected. Under the action of TdT, the ssDNA was significantly elongated, carrying numerous negative charges on its phosphate backbone, so the overall negative charge of the reporter nanoparticles was enhanced, resulting in a strong electrostatic repulsion with the ITO electrode. Simultaneously, the long ssDNA wrapped around the nanoparticles hindered the approach of the coreactant. Due to the dual effects, the ECL response of the system decreased. The constructed biosensor exhibited excellent sensitivity toward TdT over a range spanning from 1 to 100 U/L. The limit of detection is as low as 1.78 U/L. The developed approach was effectively applied to detect TdT activity in leukemic patients' leukocyte extracts.

DNA strand displacement and TdT-Mediated DNA extension for swift, convenient, and quantitative evaluation of sperm DNA integrity and its clinical implications.

DNA integrity is crucial for the clinical pregnancy outcome and offspring health, while detection methods currently used (comet assay, TUNNEL assay, SCSA, etc.) can only provide the ratio of positive sperms at the cellular level and are unable to quantitatively detect the breakpoints at the DNA molecular level. Herein, we developed a detection system based on terminal deoxynucleotidyl transferase and DNA strand displacement fluorescent probe, which could efficiently and conveniently measure the number of 3'-OH (equivalent to the number of breakpoints). We further investigated the use of this technique in assisted reproduction after completing the principle verification, system optimization, and research on analytical performance. The detection system was shown to have a good linear range from 0.01 nM to 4 nM, using single-stranded DNA with 3'-OH end as the calibrator. The system underwent thorough optimization for stability and accuracy. In comparison to the widely accepted index DFI detected by SCSA, the new system demonstrated reasonable correlation and better prediction efficiency. Its applicability was also proven through its use in assisted reproductive technology procedures.

RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues.

N6-methyladenosine (m6A) is an ubiquitous post-transcriptional modification catalyzed by METTL3/14 complex in eukaryotic mRNAs. The abnormal METTL3/14 complex activity affects multiple steps of RNA metabolism and may induce various diseases. Herein, we demonstrate the RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues. METTL3/14 complex can catalyze the methylation of RNA probe to prevent it from being cleaved by MazF. The intact RNA probe is recognized by the magnetic bead (MB)-capture probe conjugates to induce duplex-specific nuclease (DSN)-assisted cyclic digestion, exposing numerous shorter ssDNAs with 3'-OH end. The shorter ssDNAs on the MB surface can act as the primers to initiate terminal deoxynucleotidyl transferase (TdT)-enhanced tyramide signal amplification (TSA), forming the Cy5 fluorescence-encoded nanostructures. After magnetic separation, the Cy5 fluorescence-encoded nanostructures are digested by DNase I to release abundant Cy5 fluorophores that can be simply quantified by fluorescence measurement. This assay achieves good specificity and high sensitivity with a detection limit of 58.8 aM, and it can screen METTL3/14 complex inhibitors and quantify METTL3/14 complex activity at the single-cell level. Furthermore, this assay can differentiate the METTL3/14 complex level in breast cancer patient tissues and healthy volunteer tissues.

Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3'-ends of DNA. If a target protein is expressed as a TdT fusion and incubated with a DNA-encoded library (DEL) in the presence of dATP, the binders of the target induce proximity between TdT and the DNA, promoting the synthesis of a poly-adenine (polyA) tail. The polyA tail length is proportional to the binding affinity, effectively serving as a stable molecular record of binding events. The polyA tail is also a convenient handle to enrich binders with magnetic poly(dT)25 beads before sequencing. In a benchmarking system, we show that ligands spanning nanomolar to double-digit micromolar binding can be cleanly identified by TdT extension, whereas only the tightest binding ligands are identified by classical affinity selection. The method is simple to implement and can function on any DEL that bears a free 3'-end.

A highly efficient scheme for library preparation from single-stranded DNA.

Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.