Detection Of uracil-DNA Glycosylase Activity Research Articles

DNA polymerase mediated CRISPR/Cas12a trans-cleavage for dual-mode quantification of uracil DNA glycosylase activity

Since an unusual expression of uracil-DNA glycosylase (UDG) is often associated with the pathogenesis of numerous disorders, the detection of UDG activity is regarded as a promising application in disease diagnosis. Here, we develop a DNA polymerase-mediated CRISPR/Cas12a trans cleavage strategy, which can achieve dual-mode determination of UDG activity. By introducing a hairpin DNA probe containing a single uracil base, the probe undergoes specific cleavage and elongation under the existence of UDG only, thus activating the trans cleavage of ssDNA regardless of its length and sequence. To accommodate different detection modes, the ssDNA was further modified by fluorophore-quencher pairs or designed for connecting magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). Finally, the UDG activity is quantified by fluorescence signal and microparticle accumulation length on a microfluidic chip visible to the naked eye. This strategy provides a detectable minimum UDG concentration of 0.00047 U/mL for fluorescent mode and 0.0048 U/mL for microfluidic mode. Furthermore, we performed the UDG inhibition test and detected UDG activity in cell lysates, suggesting its potential for inhibitor screening and detection of UDG in biological samples.

Enzyme-Powered, Label-Free DNA Walker for Uracil-DNA Glycosylase Detection at Single-Cell Level.

Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered, label-free DNA walker was devised for UDG activity detection. Initially, a label-free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G-quadruplex sequence, enabling the generation of a label-free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme-powered walking process and generating significant dissociative G-quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label-free fluorescence response was achieved. The proposed DNA walker presented a direct and label-free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single-cell level was accomplished. Consequently, the developed DNA walker serves as a label-free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.

Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 UmL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

A novel biosensor based on tetrahedral DNA nanostructure and terminal deoxynucleotidyl transferase-assisted amplification strategy for fluorescence analysis of uracil-DNA glycosylase activity

Tetrahedral DNA nanostructure (TDN), as a classical bionanomaterial, which not only has excellent structural stability and rigidity, but also possesses high programmability due to strict base-pairs complementation, is widely used in various biosensing and bioanalysis fields. In this study, we first constructed a novel biosensor based on Uracil DNA glycosylase (UDG) -triggered collapse of TDN and terminal deoxynucleotidyl transferase (TDT)-induced insertion of copper nanoparticles (CuNPs) for fluorescence and visual analysis of UDG activity. In the presence of the target enzyme UDG, the uracil base modified on the TDN were specifically identified and removed to produce an abasic site (AP site). Endonuclease IV (Endo.IV) could cleave the AP site, making the TDN collapse and generating 3′-hydroxy (3′-OH), which were then elongated under the assistance of TDT to produce poly (T) sequences. Finally, Copper (II) sulfate (Cu2+) and l-Ascorbic acid (AA) were added to form CuNPs using poly (T) sequences as templates (T-CuNPs), resulting in a strong fluorescence signal. This method exhibited good selectivity and high sensitivity with a detection limit of 8.6 × 10−5 U/mL. Moreover, the strategy has been successfully applied to the screening of UDG inhibitors and the detection of UDG activity in complex cell lysates, which means that it has promising applications in clinical diagnosis and biomedical research.

An ultra-sensitive and specific UCBiosensor via CRISPR-Cas12a and UDG-mediated polymerase chain reaction

DNA damage is an important cause of several diseases. Uracil DNA glycosylase (UDG) is a key enzyme in DNA damage repair and used as an early diagnostic marker and a prognostic indicator for cancer. Therefore, there is an urgent need to develop a simple, low-background, ultra-sensitive UDG activity detection method for clinical applications. At present, biosensors based on CRISPR-Cas12a have become a powerful diagnostic tool owing to their high sensitivity. Here, we have established an ultra-sensitive biosensor for detecting UDG activity based on CRISPR-Cas12a coupled UDG-mediated polymerase chain reaction (U-PCR), which has the advantages of simplicity, low background, ultra-sensitivity, and specificity. We used this biosensor to multiply amplify the UDG signal, and the detection limit reached 3.5 × 10−6 U/mL, which is one of the most sensitive methods for UDG activity detection. Finally, we detected UDG activity in the lysates of cancer cells, indicating that this method could be used to detect UDG activity in real samples. The UDG inhibition test showed that the method we established is a useful tool for screening UDG inhibitors. We believe that the UDG activity detection biosensor established in this study has potential applications in biomedical research and clinical diagnosis.

Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging

As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a “double-edged sword” for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10−6 U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a.

Sensitive detection of uracil-DNA glycosylase based on AND-gate triggers

As a DNA repair enzyme, uracil-DNA glycosylase (UDG) removes uracil bases from DNA and starts the base excision repair pathway to maintain genomic integrity. It is meaningful to detect UDG activity sensitively because abnormal UDG activity is associated with cancer, immunodeficiency and many other diseases. Fluorescent detection methods have been rapidly developed because of their simplicity and sensitivity. However, in these ways, triggers used to initiate fluorescent signals only come from a single source, either the primer or the enzyme. The accidental leakage of the trigger leads to an unwanted background. To suppress the background signal further, we proposed an improved fluorescent strategy based on AND-gate triggers. In this strategy, a uracil-containing DNA oligo could inhibit the archaeal family B DNA polymerase. After UDG cleavage, the polymerase was activated (trigger-1). Meanwhile, an apurinic/apyrimidinic site was formed, then excised by Endonuclease IV, making the uracil-containing oligo become a primer with 3′-OH end (trigger-2) for further amplification. Then, an exponential amplified fluorescent signal could be generated with nicking endonuclease and well-designed templates. As the two triggers worked as an AND-gate, the leakage of any one of them was insufficient to initiate amplification reactions, resulting in low background. This AND-gate triggers based two-step amplification strategy showed a time-dependent output, with a detection limit of 1.5 × 10−4 U/mL. In addition, this solution also demonstrated a high selectivity toward UDG. Hence, this proposed strategy can be served as a promising platform for UDG activity detection and associated biomedical studies.

A structure change-induced fluorescent biosensor for uracil-DNA glycosylase activity detection based on the substrate preference of Lambda exonuclease

As one of the initiating DNA glycosylases in the base excision repair pathway, Uracil-DNA glycosylase (UDG) plays a pivotal role in maintaining genomic integrity. The abnormal expression of UDG in the organism is highly relevant to multiple diseases. Thus, rapid and sensitive detection of UDG activity is essential to aid early clinical diagnosis and biomedical research. Here we developed a rapid, sensitive and selective biosensor for UDG activity detection based on the substrate preference of Lambda exonuclease (λ exo). The protruding end in the substrate produced by UDG could be digested at a markedly high rate by λ exo, generating a detectable fluorescence signal. This proposed strategy for UDG detection exhibited high selectivity and high sensitivity (0.0001 U/mL) in a short time. It has also been successfully applied to detect UDG in real biological samples and the screening of UDG inhibitors.

A Structure Change-Induced Fluorescent Biosensor for Uracil-DNA Glycosylase Activity Detection Based on the Substrate Preference of Lambda Exonuclease

A Structure Change-Induced Fluorescent Biosensor for Uracil-DNA Glycosylase Activity Detection Based on the Substrate Preference of Lambda Exonuclease

Smart Catalyzed Hairpin Assembly-Induced DNAzyme Nanosystem for Intracellular UDG Imaging.

Uracil DNA glycosylase (UDG) is one of the key initiators for the base excision repair pathway. Since abnormal UDG expression is associated with various diseases, sensitive detection of UDG activity is critical for early clinical diagnosis. Here, a smart catalyzed hairpin assembly (CHA)-DNAzyme nanosystem is developed for intracellular UDG imaging by incorporating CHA and DNAzyme onto MnO2 nanosheets. In this strategy, the biodegradable MnO2 nanosheets are employed as nanocarriers for efficiently adsorbing and delivering five DNA probes into cells by endocytosis. Then, the MnO2 nanosheets are degraded by cellular glutathione to release the DNA modules at the same intracellular position. Liberated Mn2+, an indispensable DNAzyme cofactor, was used to promote catalytic cleavage for facilitating the cascade process in cells. Based on the uracil site-recognition and -excision operation of the target UDG, the activated CHA-DNAzyme nanosystem generates lots of DNAzyme-assisted CHA products, turning on the fluorescence resonance energy transfer response. This autocatalytic CHA-DNAzyme nanosystem provides a detectable minimum UDG concentration of 0.23 mU/mL, which is comparable to some reported UDG detection approaches. As a multiple signal amplification strategy, the CHA-DNAzyme nanosystem realizes the UDG imaging in living cells with enhanced sensitivity, indicating great promise in the prediction and diagnosis of early-stage cancer.

The dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of uracil DNA glycosylase activity

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10−5 U mL−1. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.

Single-Nanoparticle ICP-MS for Sensitive Detection of Uracil-DNA Glycosylase Activity.

Single-nanoparticle inductively coupled plasma mass spectrometry (SP-ICP-MS) has demonstrated unique advantages for the detection of biological samples. However, methods for enzyme activity detection based on SP-ICP-MS technology have been rarely explored. Here we report the development of a novel SP-ICP-MS assay for uracil-DNA glycosylase (UDG) activity detection based on its ability to specifically recognize and remove uracil to induce the cleavage of the DNA probe. Our design allows the generation of single gold nanoparticles correlated to the specific enzymatic reaction for a highly sensitive SP-ICP-MS measurement. The developed assay enables sensitive UDG activity detection with a detection limit of 0.0003 U/mL. The cell lysate analysis by the developed assay reveals its applicability for the detection of UDG activity in real samples. It is envisioned that our design may provide a new paradigm for developing the SP-ICP-MS assay for enzyme activity detection in biological samples.

Distance-Based Biosensor for Ultrasensitive Detection of Uracil-DNA Glycosylase Using Membrane Filtration of DNA Hydrogel.

In the deoxyribonucleic acid (DNA) repair pathways, DNA repair enzymes have great significance for genomic integrity. As one important initiator of the base-excision repair pathway, the aberrant activity of uracil-DNA glycosylase (UDG) is closely associated with many diseases. Herein, we developed a simple distance-based device for visual detection of UDG activity using a load-free DNA hydrogel. The DNA hydrogel consists of polyacrylamide-DNA chains being bridged by a single-stranded DNA crosslinker containing a responsive uracil base site. UDG can recognize and remove the uracil, resulting in the cleavage effect of the DNA crosslinker strand with the assistance of endonuclease IV (Endo IV). Plugging one end of the capillary tube, the DNA hydrogel acting as a filter membrane separator would control molecules to flow into the tube. The integrity of the DNA hydrogel networks is affected by the excision of UDG. Therefore, taking full advantage of membrane filtration of the DNA hydrogel, the activity of UDG can be quantitatively detected via reading the distance of the red indicator solution in the capillary tube. Without any instruments and complicated procedures, this method realizes high sensitivity and specificity for the detection of UDG as low as 0.02 mU/mL and can even measure UDG in complex cell samples. Additionally, this method is simple, universal, and can be used to screen inhibitors, which shows great potential for point-of-care testing, clinical diagnosis, and drug discovery.

A DNAzyme-driven random biped DNA walking nanomachine for sensitive detection of uracil-DNA glycosylase activity.

Highly specific and ultrasensitive detection of uracil-DNA glycosylase (UDG) activity is of great significance for maintaining genomic integrity and medical research of related diseases. Here, we constructed a random DNA walking nanomachine based on a DNAzyme for UDG activity detection on the AuNP (Au nanoparticle) surface. When UDG is present, the U bases in the Y structure are removed, resulting in AP sites, which will be cleaved by Endo-IV to generate a 3' concave end for Exo-III, causing the locking strand of the DNAzyme to be completely hydrolyzed by the Exo-III and release the walking strand to randomly pair with the substrate strand on the AuNP surface; then, the walking strand exerts its cleavage activity with the assistance of Mg2+ to cleave the substrate strand and keep the fluorophore 6-carboxyfluorescein (FAM) away from the surface of the AuNP, which restores the fluorescence signal of this system. In this way, sensitive detection of UDG can be realized, and the detection limit is as low as 3.69 × 10-6 U mL-1. In addition, we found that this method is highly specific to UDG and can be used to detect UDG specifically in complex samples, which has certain application prospects in biomedical research and clinical diagnosis related to UDG.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10−6 U/mL and 1 × 10−4 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.

One-step G-quadruplex-based fluorescence resonance energy transfer sensing method for ratiometric detection of uracil-DNA glycosylase activity

Uracil-DNA glycosylase (UDG) is a crucial enzyme in base excision repair (BER) pathway. It can repair the uracil-induced DNA lesions and maintain the integrity of genome. In this paper, we developed a facile and ratiometric strategy for UDG activity detection using fluorescence resonance energy transfer (FRET). One double-stranded DNA (dsDNA) substrate consisting of strand 1 (dual-fluorescent dye-modified G-quadruplex sequence single-stranded DNA (ssDNA)), carboxyfluorescein (FAM) acted as donor and tetramethylrhodamine (TAMRA) as acceptor) and strand 2 (the complementary sequence of strand 1 containing three mismatched bases and three uracil bases) was introduced. When the UDG-catalyzed uracil is removed from dsDNA, the thermo-stability of dsDNA is decreased and the dual-fluorescent dye-modified G-quadruplex sequence ssDNA is released. Then, the ssDNA transforms into a G-quadruplex comformation, which brings the labeled FAM and TAMRA into close proximity, resulting in a strong FRET signal. In the absence of UDG, the relatively stable dsDNA separates the labeled FAM and TAMRA, giving a weak FRET signal. Thus, by measuring the system fluorescence intensity and exploiting FRET signal difference, UDG activity can be detected in a simple process. The detection limit is 0.087 U/mL without requiring additional signal amplification process. Besides, our developed strategy can also be used for screening the UDG inhibitors in a ratiometric fluorescence detection way.

A Methodology for Ultrasensitive Detection of Sequence-Specific DNA or Uracil-DNA Glycosylase Activity.

Ultrasensitive detection of sequence-specific DNA and uracil-DNA glycosylase (UDG) activity shows great practical significance in clinical diagnostic and biomedical studies. Here, a methodology based on a CRISPR/Cas12a system coupled with enhanced strand displacement amplification (E-SDA) was innovatively established for sequence-specific DNA or UDG activity detection. Sequence-specific DNA or DNA primers processed by UDG and Endonuclease IV can initiate E-SDA, generating auxiliary DNA chains, which act as activators to unlock the indiscriminate collateral cleavage activities (trans-cleavage) of the CRISPR/Cas12a. Then, the activated CRISPR/Cas12a, which intrinsically possesses the ability of significant signal amplification, can indiscriminately cleave the added cleavage reporters in the system. Thus, the multistep amplification of the method was obtained. Under the selected experimental conditions, the established method can achieve an actual sensitivity of sequence-specific DNA up to 100 aM within 2.5 h or ultralow UDG activity (3.1×10-5 U/mL) detection within 3.5 h. We believe that the proposed method will have great potential for practical application in ultrasensitive detection of sequence-specific DNA or UDG activity.

Target-triggered activation of rolling circle amplification for label-free and sensitive fluorescent uracil-DNA glycosylase activity detection and inhibition

The expression variations of uracil-DNA glycosylase (UDG) can be used as effective biomarkers for the evaluation of gene regulation and related diseases. Here, by using a new target-triggered activation of rolling circle amplification (RCA) signal enhancement strategy, we have established a sensitive and label-free fluorescent approach for UDG activity detection and inhibition. The target UDG specifically recognizes and excises the uracil bases in a three-strand containing DNA complex to liberate one of the strands. Subsequent ligation of the excised DNA complex converts it into a suitable primer/circular template structure for the initiation of RCA for the generation of long DNA sequences with many repeated G-quadruplexes. Protoporphyrin IX further binds these G-quadruplexes to show substantially enhanced fluorescence to achieve sensitive detecting the activity of UDG with the detection limit as low as 0.00014 U mL−1. Besides, this assay approach has a high specificity toward UDG and can also be utilized to evaluate its inhibition by the uracil glycosylase inhibitor, highlighting the promising applications for convenient and sensitive UDG activity detection and inhibition for disease diagnosis and drug screening.

Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification.

Sensitive detection of uracil-DNA glycosylase (UDG) activity is very important in the study of many fundamental biochemical processes and clinical applications. Here, we develop a novel assay for the detection of UDG activity by using the self-initiating multiple rolling circle amplification (SM-RCA) strategy. We first design a trigger probe modified with NH2 at its 3′-terminal and uracil base in the middle of sequence, which is complementary to a cyclized padlock probe. In the presence of UDG, a uracil base can be excised by UDG to generate an apurinic/apyrimidinic (AP) site. The AP site is recognized and cleaved by endonuclease IV (Endo IV), releasing the primer with 3′-OH. The primer can trigger the rolling circle amplification (RCA) reaction, producing a long and repeated DNA strand embedded some uracil bases. These uracil bases can be cleaved by UDG and Endo IV again, and then, more primers are generated to initiate SM-RCA reaction, producing large amounts of DNA product. Afterward, the DNA product is measured by a specific DNA fluorescence dye for quantitative detection of UDG activity. The linear range of the method is 5 × 10–5 to 1.25 × 10–3 U/mL, and the detection limit is 1.7 × 10–5 U/mL. This method not only utilizes the target UDG itself to trigger RCA but also further induces SM-RCA reaction, providing a simple, sensitive, and cost-effective strategy for the detection of glycosylase and clinical diagnosis.