Terminal Deoxynucleotidyl Transferase Activity Research Articles

A strand-elongation initiated DNAzyme walker for terminal deoxynucleotidyl transferase activity detection

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase found to be overactive in most leukemia cells and is recognized as a biomarker of leukemia. Here, we develop a strand-elongation initiated DNAzyme walker to detect TdT activity. The walker is constructed by modifying specially designed track strand (TS) and incomplete walking strand (WS) onto the surface of a mesoporous silica nanoparticle (MSN) loaded with large amounts of rhodamine 6 G (Rh6 G) in its pores. The TS is a hairpin DNA with an RNA cleavage site, serving as substrate for DNAzyme and encapsulating the Rh6 G. The incomplete WS contains one side of binding arm and partial catalytic core of DNAzyme. Under the action of TdT, the incomplete WS is elongated with polyA, completing the lacking part of catalytic core and the other side of binding arm of DNAzyme. Then the completed DNAzyme cleave the TSs, unblocking and releasing the Rh6 G to indicate the TdT activity. This walker is completed and initiated only after the action of TdT, ensuring its strict specificity for TdT activity. Besides, each WS cleave multiple TSs, and each cleavage leads massive Rh6 G to be released. Such double-amplified signal accumulation endows the walker with good sensitivity. This walker specifically detected TdT activity down to 0.093 U mL−1, and successfully detected TdT activity in human serum. The proposed walker demonstrates a novel method for specific and sensitive detection of TdT activity, providing an effective tool for TdT-related biological research and leukemia theranostics.

A cathode photoelectrochemical assay of terminal deoxynucleotidyl transferase activity based on Ag-AgI-CNTs composite and surface multisite strand displacement amplification

Photocathode-based assay is anti-interference for real sample detection. Photocathode produces low photocurrent signal and gives rise to poor sensitivity. Herein, a novel cathode photoelectrochemical (CPEC) sensing platform based on Ag-AgI-CNTs as photocathode material and K3[Fe(CN)6] as photoelectron acceptor was established. Since [Fe(CN)6]3- effectively accepted photoelectrons from Ag-AgI-CNTs, it greatly enhanced the CPEC response. Combining a surface multisite strand displacement amplification (SMSDA) strategy, the CPEC platform was applied for the activity assay of terminal deoxynucleotidyl transferase (TdT). In this proposal, oligo dT primer tethered on CPEC platform was in-situ extended to generate a polyA tail. Then the polyA tail formed a stable multi-point hybrid structure with the adjacent oligo dT. After launching the SMSDA, the CPEC platform was covered by more elongated polynucleotide chains and network, which acutely hampered the photoelectron transfer (eT) between photocathode and electron acceptor and caused a reduced photocurrent. The CPEC sensor possessed a satisfactory linear response from 6×10-5-0.1 U and a low detection limit of 1.1×10-5 U. The strategy offered a more specific and sensitive method for TdT activity assay. It was feasible in the field of TdT-based biochemical research, drug screening, and disease diagnosis.

Self-assembly of a polythymine embedded activatable molecular beacon for one-step quantification of terminal deoxynucleotidyl transferase activity

We describe an isothermal, single-reaction, and one-step method for signal-on quantification of terminal deoxynucleotidyl transferase (TdT) activity based on the periodic elongation and assembly of polythymine embedded activatable molecular beacon (PTA-MB) into DNA nanostructures. PTA-MB is easily designed according to the rule of the conventional molecular beacon (MB) but engineered with a polyT composed loop. Upon exposure to the specific target TdT, the MB is first elongated with an adenine-rich (A-rich) long chain so that it can then act as the anchoring substrate to capture many original PTA-MBs along its strand. Their unfolding contributes to preliminary fluorescence emission. Significantly, the assembled PTA-MBs can also be elongated and hybridized with residual free PTA-MBs for the second round of signal amplification. Accordingly, multiple rounds of elongation, assembly, and activation of initial PTA-MBs can lead to the formation of DNA nanostructures and induce a dramatically enhanced fluorescence signal for qualitative and quantitative evaluation of TdT activity. The final assay indicated a limit of detection (LOD) of 0.042 U mL−1 TdT and showed excellent selectivity for TdT versus other common enzymes. Moreover, the practical applicability was validated by direct/absolute quantification of TdT in real biological specimens and accurate monitoring of the activity of TdT pretreated by low/high temperature and heavy metal ions. These findings demonstrated that this functional PTA-MB and its unique assembly behavior is most likely to promote the study of oligonucleotide probe-based DNA assembly, providing a reliable, convenient, and universal platform for precise and point-of-care monitoring of various biomolecules.

125I seeds irradiation inhibits tumor growth and induces apoptosis by Ki-67, P21, survivin, livin and caspase-9 expression in lung carcinoma xenografts

BackgroundLung cancer is a fatal disease and a serious health problem worldwide. Patients are usually diagnosed at an advanced stage, and the effectiveness of chemotherapy for such patients is very limited. Iodine 125 seed (125I) irradiation can be used as an important adjuvant treatment for lung carcinoma. The purpose of this study was to examine the role of irradiation by 125I seeds in human lung cancer xenograft model and to determine the underlying mechanisms involved, with a focus on apoptosis.Methods40 mice with A549 lung adenocarcinoma xenografts were randomly divided into 4 groups: control group (n = 10), sham seed (0 mCi) implant group (n = 10), 125I seed (0.6 mCi) implant group (n = 10) and 125I seed (0.8 mCi) implant group (n = 10), respectively. The body weight and tumor volume, were recorded every 4 days until the end of the study. Apoptotic cells were checked by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and activities of caspase-3 and caspase-8 enzyme were tested. Expression of P21, survivin, livin, caspase-9 and proliferating cell nuclear antigen (Ki-67) was detected with immunohistochemical staining.ResultsThe results of TUNEL staining assays showed that 125I seed irradiation suppresses the growth of lung cancer xenografts in nude mice and induced apoptosis. The activity of caspase-3 and caspase-8 was significantly higher. The expression levels Ki67, survivin and livin were substantially downregulated, while P21 and caspase-9 protein expression were significantly increased following 125I seed irradiation. This study revealed that 125I seed irradiation could significantly change apoptosis-related protein in human lung cancer xenografts.ConclusionsOverall, our study demonstrates that radiation exposure by 125I seeds could be a new treatment option for lung cancer.

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

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.

A terminal extension-actuated isothermal exponential amplification strategy toward the ultrasensitive and versatile detection of enzyme activity in a single cell

Terminal deoxynucleotidyl transferase (TdT) plays an important role in regulating a wide range of genomic processes. The sensitive and accurate detection of cellular TdT activity, particularly at the single-cell level, is highly significant for leukemia-associated biomedical and biological studies. Nevertheless, owing to the limited sensitivity of the existing TdT assays, the quantification of TdT activity at the single-cell level remains a big challenge. Herein, a simple but ultrasensitive method for assaying TdT activity is proposed based on terminal extension actuated loop-mediated isothermal amplification (TEA-LAMP). By using the TdT-induced extension product as an actuator, TdT activity is amplified twice by terminal extension and LAMP in an exponential manner and finally converted to a remarkably amplified fluorescent signal. In this study, as low as 2 × 10−8 U/μL TdT can be clearly detectable with the elegant TEA-LAMP strategy. Such an ultrahigh sensitivity enables the direct determination of TdT activity in individual single cells. In the meantime, by employing TdT as a co-factor, this strategy can also be applied to detecting other enzymes that can catalyze the DNA terminal hydroxylation. This work not only reports the up-to-now most sensitive TdT detection strategy at a single-cell level but also opens the new gate for versatile enzyme activity detection.

Molecular roulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT

AbstractNucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1 is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1 mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1 mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1 mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P < .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863_864 over 4 orders of magnitude (ρ = 0.484, P < .0001). Children have a different NPM1 mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity.

Terminal deoxynucleotidyl transferase promotes acute myeloid leukemia by priming FLT3-ITD replication slippage

AbstractFLT3–internal tandem duplications (FLT3-ITDs) are prognostic driver mutations found in acute myeloid leukemia (AML). Although these short duplications occur in 25% of AML patients, little is known about the molecular mechanism underlying their formation. Understanding the origin of FLT3-ITDs would advance our understanding of the genesis of AML. We analyzed the sequence and molecular anatomy of 300 FLT3-ITDs to address this issue, including 114 ITDs with additional nucleotides of unknown origin located between the 2 copies of the repeat. We observed anatomy consistent with replication slippage, but could only identify the germline microhomology (1-6 bp) anticipated to prime such slippage in one-third of FLT3-ITDs. We explain the paradox of the “missing” microhomology in the majority of FLT3-ITDs through occult microhomology: specifically, by priming through use of nontemplated nucleotides (N-nucleotides) added by terminal deoxynucleotidyl transferase (TdT). We suggest that TdT-mediated nucleotide addition in excess of that required for priming creates N-regions at the duplication junctions, explaining the additional nucleotides observed at this position. FLT3-ITD N-regions have a G/C content (66.9%), dinucleotide composition (P < .001), and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0; P = .0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. Although the physiological role of TdT is to increase antigenic diversity through N-nucleotide addition during V(D)J recombination of IG/TCR genes, here we propose that illegitimate TdT activity makes a significant contribution to the genesis of AML.

Single Primer Based Multisite Strand Displacement Reaction Amplification Strategy for Rapid Detection of Terminal Deoxynucleotidyl Transferase Activity.

A fluorescence-based multisite strand displacement reaction (MSSDR) amplification strategy is developed for the rapid, sensitive, and selective detection the activity of terminal deoxynucleotidyl transferase (TdT). Oligo dT primer was used for the TdT extension reaction, then the left oligo dT primers were hybridized to the TdT extension reaction product by end to end tiled style and initiated the MSSDR by Klenow polymerase, subsequently, 3' terminals of these single-strand DNA produced by MSSDR are folded back to complement themselves with the adjacent sequences, and Klenow polymerase makes it into double-stranded DNA (dsDNA). The final dsDNA products were analyzed via dsDNA specific fluorescent dye. This method enables rapid (less than 100 min) and sensitive (limit of detection, LOD, 1.35 × 10-5 U) detection and has been demonstrated to work well using a real biosample. Our design would not only serve as a new prototype for high-throughput automated analysis and clinic diagnostic application but also has promising potential for improving the sensitivity of those TDT related biosensing system.

Visual Detection of Cucumber Green Mottle Mosaic Virus Based on Terminal Deoxynucleotidyl Transferase Coupled with DNAzymes Amplification.

A simple, rapid, and sensitive visual detection method for observing cucumber green mottle mosaic virus was reported based on the template-independent polymerization activity of terminal deoxynucleotidyl transferase (TdT), coupled with the cascade amplification of Mg2+-dependent DNAzyme and hemin/G-quadruplex DNAzyme. Briefly, the hybridized dsDNA of T1/P1 was cut into two parts at its position of 5′-AA↓CG↑TT-3′ by the restricted enzyme AcII. The longer, newborn fragment originating from P1 was tailed at its 3’-end by oligo dG, and an intact enzymatic sequence of Mg2+-dependent DNAzyme was generated. The substrate sequence in the loop segment of the hairpin probe (HP) hybridized with the newborn enzymatic sequence and was cleaved into two parts in the presence of Mg2+. The locked G-quadruplex sequence in the stem segment of the HP was released, which catalyzed the oxidation of ABTS2- in the presence of H2O2, and the resulting solution turned green. A correlation between the absorbance and concentration of T1 was obtained in a range from 0.1 pM to 2 nM, with a detection limit of 0.1 pM. In addition to promoting a lower detection limit and shorter monitoring time, this method also demonstrated an excellent selectivity to single or double nucleotide changes. Therefore, the designed strategy provided a rapid and efficient platform for viral inspection and plant protection.

Fluorometric determination of the activity of the biomarker terminal deoxynucleotidyl transferase via the enhancement of the fluorescence of silver nanoclusters by in-situ grown DNA tails.

The activity of terminal deoxynucleotidyl transferase (TdTase) is a biomarker for routine diagnosis of acute leukemia. A method has been developed for the determination of TdTase activity. It is based on the use of silver nanoclusters (AgNCs) whose yellow fluorescence is enhanced by an in-situ grown DNA tail of TdTase-polymerized and guanine-rich DNA at the 3' end of a hairpin DNA. The fluorescence, best measured at excitation/emission peaks of 530/585nm, increases linearly in the 1 to 35mUmL-1 TdTase activity range. The detection limit is 0.8mUmL-1. The method is cost-efficient, selective and convenient. It integrates enhancement of the fluorescence of AgNCs and target recognition into a single process. Graphical abstract Schematic presentation of a method for determination of TdTase activity. It is based on AgNCs fluorescence enhanced by in-situ grown TdTase-polymerized G-rich DNA tail. The method integrates AgNCs fluorescence enhancement and the target recognition into a single process.

Surface-functionalized MoS2 ultrathin nanosheets for electrochemical monitoring terminal deoxynucleotidyl transferase activity based on in-situ polymerized DNA

An efficient electrochemical biosensing strategy for terminal deoxynucleotidyl transferase (TdT) activity is developed based on thionin functionalized MoS2 ultrathin nanosheets (Th/MoS2 UTNS) and TdT-induced DNA polymerization. MoS2 ultrathin nanosheets is firstly achieved via a facile pyrolytic approach assisted by g-C3N4 sacrificial template. Followed by subsequent surface-functionalization with thionin, the Th/MoS2 UTNS composite is obtained and designed as a sensing platform for TdT activity detection. In this approach, TdT catalyzes the polymerization of deoxyribonucleotides triphosphate (dNTP) substrate of 50% dATP-50% dTTP to the 3′-OH terminus of DNA primer and in-situ produces dsDNA consequently, which can be strongly immobilized onto thionin by intercalation and electrostatic interaction, resulting in a decreased redox signal of thionin. The proposed sensor exhibits a wide linear response for TdT in the range from 0.01 U to 2.44 U with a low detection limit of 0.0025 U. By elucidating an efficient biosensing platform on the basis of surface engineered MoS2 ultrathin nanosheets, this work sheds light on the development of novel strategy for detecting other nucleic acid or protein analytes.

Label-free and sensitive detection assay for terminal deoxynucleotidyl transferase via polyadenosine-coralyne fluorescence enhancement strategy

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.

Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H2O2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

In situ grown DNA nanotail-templated silver nanoclusters enabling label-free electrochemical sensing of terminal deoxynucleotidyl transferase activity

A novel label-free electrochemical strategy was established based on the unique electro-catalytic activity of graphene oxide (GO)-supported terminal deoxynucleotidyl transferase (TdT)-generated C-rich DNA nanotail-templated silver nanoclusters (DNA-AgNCs). TdT can catalyze the deoxycytidine triphosphate (dCTP) to the 3′-OH terminus of single-stranded DNA (ssDNA) with no template; then, in the presence of Ag(I), TdT-generated C-rich DNA sequence was employed for the synthetic template of AgNCs because of the formed complexes of nitrogen atoms of cytosine based with silver atoms. We proved that in situ grown DNA nanotail-templated AgNCs can be adsorbed on GO-modified electrode and possess high electro-catalytic activity to H2O2 reduction, presenting a good electrochemical indicator for signal readout. Under optimal conditions, the proposed biosensor could be employed for quantitatively monitoring TdT activity and within a dynamic range from 0.4 to 90U/mL and a low limit of detection is 0.08U/mL. With high sensitivity and excellent selectivity, this strategy offers a facile, convenient and specific electrochemical method for TdT activity detection and its relevant inhibitors screening. It holds a promising potential in the practical application of TdT-based biochemical research, disease diagnosis and drug discovery.

Fluorometric determination of the activity and inhibition of terminal deoxynucleotidyl transferase via in-situ formation of copper nanoclusters using enzymatically generated DNA as template

The authors report on a simple strategy for sensitive determination of the activity of terminal deoxynucleotidyl transferase (TdT) using copper nanoclusters (CuNCs) as fluorescent probes. TdT-polymerized long chain AT-rich DNA serves as a template for the synthesis of the CuNCs, and TdT activity is detected fluorometrically at excitation/emission wavelengths of 340/570 nm. The protocol relies on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs. The calibration plot is linear in the 0.7 to 14 U L−1 activity range, with a 60 mU L−1 detection limit (at a signal-to-noise ratio of 3). The protocol was applied to determine TdT activity in acute lymphatic leukemia cells. This approach is selective, simple, convenient and cost-efficient because a complex DNA sequence is not required. In our perception, the method provides a viable new platform for monitoring the activity and inhibition of TdT.

Single quantum dot-based nanosensor for rapid and sensitive detection of terminal deoxynucleotidyl transferase.

We developed a simple and rapid method for terminal deoxynucleotidyl transferase (TdT) assay on the basis of the polymerization-directed exonuclease-assisted construction of a single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor. This method is very sensitive with a detection limit as low as 1 × 10-6 U μL-1, and it can be used for the screening of TDT inhibitors and accurate quantification of TdT activity even in 5 cancer cells.

Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination

Eukaryotic DNA polymerase of the polX family, such as pol μ and terminal deoxynucleotidyl transferase (TdT), are key components of the non-homologous end-joining or V(D)J recombination machinery, respectively. The established role of TdT is to add random nucleotides during V(D)J recombination. Here we show that TdT also has a templated-polymerase activity, similar to pol μ, in the presence of higher concentrations of a downstream DNA duplex, and performs a micro-homology single base-pair search to align the DNA synapsis. To understand the molecular basis of this alignment, we solve crystal structures of TdT with four DNA strands and study the influence ofthe 3' protruding end. Two mutations in TdT inspired by sequence alignments with pol μ further improve the templated activity. We propose that both templated and untemplated activities of TdT are needed to explain the distributions of lengths of N regions observed experimentally in Tcell receptors and antibodies.

Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

DNA methylation, catalyzed by DNA methyltransferases (MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein (ScGFP)/graphene oxide (GO) interaction, coupled with methylation-initiated template- free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3'-terminal (DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase (TdT), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with ScGFP and form the ScGFP/DNA nanocomplex. Such nanocomplex can effectively protect ScGFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of ScGFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of ScGFP/GO. The method was successfully used to detect the activity of DNA adenine methylation (Dam) MTase with a wide linear range (0.1–100 U/mL) and a low detection limit of 0.1 U/mL. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect TdT activity.

High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire.

The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals.Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR.Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides.Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination.