Single-stranded pre-methylated 5mC adapters uncover the methylation profile of plasma ultrashort Single-stranded cell-free DNA.

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Plasmoelectronic-Based Ultrasensitive Assay of Tumor Suppressor microRNAs Directly in Patient Plasma: Design of Highly Specific Early Cancer Diagnostic Technology.

It is becoming understood that microRNAs hold great promise for noninvasive liquid biopsies for screening for different types of cancer, but current state-of-the-art RT-PCR and microarray techniques have sensitivity limitations that currently restrict their use. Herein, we report a new transduction mechanism involving delocalization of photoexcited conduction electrons wave function of gold triangular nanoprism (Au TNP) in the presence of -ssDNA/microRNA duplexes. This plasmoelectronic effect increases the electronic dimension of Au TNPs and substantially affects their localized surface plasmon resonance (LSPR) properties that together allow us to achieve a sensitivity for microRNA assay as low as 140 zeptomolar concentrations for our nanoplasmonic sensors. We show that the position of a single base-pair mismatch in the -ssDNA/microRNA duplex dramatically alters the LSPR properties and detection sensitivity. The unprecedentedly high sensitivity of nanoplasmonic sensors has allowed us to assay four different microRNAs (microRNA-10b, -182, -143, and -145) from bladder cancer patient plasma (50 µL/sample). For the first time, we demonstrate the utility of a label-free, nanoplasmonic sensor in quantification of tumor suppressor microRNAs, the level of tumor suppressor microRNAs goes down in a cancer patient as compared to normal healthy individuals, in metastatic and nonmetastatic bladder cancer patient plasma. Our statistical analysis of patient samples unequivocally suggests that the tumor suppressor microRNAs are more specific biomarkers ( p-value of <0.0001) than oncogenic microRNAs for differentiation between metastatic and nonmetastatic bladder cancer, and nonmetastatic cancer from healthy individuals. This work demonstrating the electron wave functions delocalization dependent ultrasensitive LSPR properties of noble metal nanoparticles has a great potential for fabrication of miniaturized and extremely powerful sensors to investigate microRNA properties in other cancers (for example breast, lung, and pancreatic) through liquid biopsy.

Colorimetric determination of cytosine-rich ssDNA by silver(I)-modulated glucose oxidase-catalyzed growth of gold nanoparticles.

A colorimetric assay is described for determination of cytosine-rich ssDNA at physiological pH values. The working principle is based on (a) Ag(I) ion-induced formation of an i-motif structure, and (b) glucose oxidase-controlled growth of gold nanoparticles (AuNPs). The combination between Ag+ and cytosine-rich DNA can modulate the generation of H2O2 resulting from enzyme catalyzed glucose oxidation. Depending on the amount of H2O2 formed, the solution containing the AuNPs will turn red in the presence of cytosine-rich ssDNA but blue in the absence of such DNA if Ag+ is added before the formation of the red AuNPs. Upon addition of C-DNA at different concentrations, the peak shift (Δλ) of the AuNP solution relative to the SPR peak position (560 nm) in the absence of C-DNA is taken as the signal readout. The method shows a good linear response toward C-DNA over the range 10-200 nM with a detection limit of 2.7 nM. It may also be performed visually. The photometric assay is highly sensitive, specific, and rapid. The method is particularly attractive in terms of applications such as in human serum analysis, a colorimetric logic gate, and the calculation of binding constants for the interaction between Ag+ and glucose oxidase (GOx), and between Ag+ and cytosine-rich ssDNAs. Graphical abstract Schematic presentation of colorimetric detection of cytosine (C)-rich ssDNA (C-DNA) based on the modulation of the glucose oxidase (GOx)-catalyzed growth of gold nanoparticles (AuNPs) with Ag+ as the enzyme inhibitor.

Fluorometric determination of ssDNA based on functionalized magnetic microparticles and DNA supersandwich self-assemblies.

A method is described for the determination of DNA via nucleic acid amplification by using nucleic acid concatemers that result from DNA supersandwich self-assemblies (SSAs). The method employs two auxiliary probes to form self-assembled biotin SSAs. These exhibit strong fluorescence if labeled with intercalator SYBR Green I. In the presence of the target (as exemplified for a 30-mer), streptavidin is released from the surface of the functionalized magnetic microparticles (FMMPs) by competitive hybridization on the surface. However, the SSA products do not conjugate to the FMMPs. This leads to a large amount of SYBR Green I intercalated into the concatemers and eventually results in amplified fluorescence in the supernate. The SSA products can be prepared beforehand, and amplification therefore can be completed within 50 min. The method is more efficient than any other conventional amplification. The detection limit for the 30-mer is 26.4 fM which is better by a factor of 10 compared to other amplification methods. Conceivably, the method can be further extended to the determination of a wide variety of targets simply by replacing the sequences of the probes. Finally, this rapid and highly sensitive method was employed for detection of Ebola virus gene (≈30-mer) and ATP in spiked serum with satisfactory results. Graphical abstract A high sensitivity and efficiency bioassay is described based on functionalized magnetic microparticles and DNA supersandwich self-assemblies.

Screening of specific nucleic acid aptamers binding tumor markers in the serum of the lung cancer patients and identification of their activities.

Lung cancer is by far the leading cause of cancer death in the world. Despite the improvements in diagnostic methods, the status of early detection was not achieved. So, a new diagnostic method is needed. The aim of this study is to obtain the highly specific nucleic acid aptamers with strong affinity to tumor markers in the serum of the lung cancer patients for targeting the serum. Aptamers specifically binding to tumor markers in the serum of the lung cancer patients were screened from the random single-stranded DNA library with agarose beads as supports and the serum as a target by target-substituting subtractive SELEX technique and real-time quantitative polymerase chain reaction technique. Subsequently, the secondary single-stranded DNA library obtained by 10 rounds of screening was amplified to double-stranded DNA, followed by high-throughput genome sequence analysis to screen aptamers with specific affinity to tumor markers in the serum of the lung cancer patients. Finally, six aptamers obtained by 10 rounds of screening were identified with high specific affinity to tumor markers in the serum of the lung cancer patients. Compared with other five aptamers, the aptamer 43 was identified both with the highest specificity to bind target molecule and without any obvious affinity to non-specific proteins. The screened aptamers have relatively high specificity to combine tumor markers in the serum of the lung cancer patients, which provides breakthrough points for early diagnosis and treatment of lung cancer.

Frequency shift Raman-based sensing of serum MicroRNA for ultrasensitive cervical cancer diagnosis.

Cervical cancer is the most common gynaecological tumor. The development of a sensor for the ultrasensitive detection of cervical cancer is significant in guaranteeing its prognosis. Herein, we proposed a novel surface-enhanced Raman scattering (SERS) analysis platform using a frequency shifts-based sensing model for rapid and ultrasensitive microRNA (miRNA) assay. During the analysis process, miR-21 can be captured by the single-stranded DNA (ssDNA) modified on the platform which is complementary pairing with miR-21. The connection of miR-21 can lead to the variation of the molecular weight and result in the deformation extent of the Raman report molecule 6Thioguanine (6TG); thus, the peak at 1301 cm-1 due to the ring C-N stretches of 6TG shifts to lower frequency. The detection limit (LOD) of the proposed SERS analysis platform is as low as 8.32 aM. Moreover, the platform also has excellent specificity and repeatability, with the relative standard deviation (RSD) value of 6.53 %. Serum samples of cervical cancer patients and healthy subjects were analyzed via the platform and the accuracy of the detection results was verified by qRT-PCR, revealing that SERS results and qRT-PCR results have high homogeneity. Thus, the platform can serve as a potential tool for clinical diagnosis of cervical cancer.

Dielectrophoretic isolation of DNA and nanoparticles from blood.

The ability to effectively detect disease-related DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for viable diagnostics and therapy monitoring. A DEP method has been developed which allows the rapid isolation, concentration and detection of DNA and nanoparticles directly from human and rat whole blood. Using a microarray device operating at 20 V peak-to-peak and 10 kHz, a wide range of high molecular weight (HMW)-DNA and nanoparticles were concentrated into high-field regions by positive DEP, while the blood cells were concentrated into the low-field regions by negative DEP. A simple fluidic wash removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high-field regions where they can be detected by fluorescence. HMW-DNA could be detected at 260 ng/mL, which is a detection level suitable for analysis of disease-related cell-free circulating DNA biomarkers. Fluorescent 40 nm nanoparticles could be detected at 9.5 × 10(9) particles/mL, which is a level suitable for monitoring drug delivery nanoparticles. The ability to rapidly isolate and detect DNA biomarkers and nanoparticles from undiluted whole blood will benefit many diagnostic applications by significantly reducing sample preparation time and complexity.

Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics.

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

Methods for the Detection of Circulating Pseudogenes and Their Use as Cancer Biomarkers.

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.

Kinetics and mechanisms for removal of circulating single-stranded DNA in mice.

Clearance of exogenous ssDNA from circulation was rapid and occurred primarily through the liver. With higher doses of single-stranded DNA (ssDNA), both liver uptake and whole blood clearance approached a maximum, enabling larger amounts of ssDNA to persist in the circulation. The large molecular weight material (precipitable ssDNA) which remained in circulation was rapidly cleaved to 20,000-30,000 mol wt fragments by endonucleases, at least some of which could be demonstrated in plasma in vitro. Mononucleotide breakdown products appeared rapidly in circulation with no lag phase, suggesting that exonuclease activity was not dependent upon prior phagocytosis. Since no exonuclease activity could be demonstrated in plasma in vitro, it was postulated that breakdown of ssDNA by exonucleases occurs on the surface of hepatocytes of Kupffer cells.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome.

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

The Ratio of ssDNA to dsDNA in Circulating Cell-Free DNA Extract is a Stable Indicator for Diagnosis of Gastric Cancer.

Due to the different mechanisms of cell-free DNA production, the single-stranded DNA to double-stranded DNA ratio in blood maybe different between healthy individuals and gastric cancer (GC) patients. We aimed to explore the potential application of this ratio in GC diagnosis. The plasma cell-free DNA extracts from 118 healthy individuals and 106 GC patients were prepared. The levels of single-stranded DNA or double-stranded DNA in plasma, and the single-stranded DNA to double-stranded DNA ratio on the diagnostic efficiency for GC were assessed with ROC curve. The relationships between this ratio and the clinical characteristics of GC patients were analyzed. The ratios in 63 GC patients before and after surgery were compared. In healthy individuals, the single-stranded DNA to double-stranded DNA ratio was not affected by factors including age, gender and BMI, and subjected to normal distribution (P = 0.1090). GC patients had a lower value of this ratio than healthy individuals (P < 0.0001). Considering this ratio as a GC diagnostic indicator, the area under ROC curve (AUC) was 0.923[95% confidence interval (CI):0.880-0.955]. This ratio in unresectable GC was obviously lower than that in resectable GC (P = 0.0045). There was a rank correlation between this ratio and GC TNM staging (rho = -0.266, P = 0.0058), but it had no correlation with tumor size (r = 0.14, P = 0.145). Additionally, this ratio was not affected by hemolysis and repeated freeze-thaw of blood samples, and was significantly elevated after surgery(P < 0.0001). The single-stranded DNA to double-stranded DNA ratio in plasma is a stable non-invasive indicator for GC diagnosis.

Electric Field-Induced Release and Measurement (EFIRM): Characterization and Technical Validation of a Novel Liquid Biopsy Platform in Plasma and Saliva.

Electric field-induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non-small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma.

Flap endonuclease-initiated enzymatic repairing amplification for ultrasensitive detection of target nucleic acids.

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 µL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.

Prozone-like phenomenon found in chemiluminescent enzyme immunoassay using magnetic particles for measurement of serum anti-single stranded DNA antibody titers: Definition and management.

BACKGROUND: Serum anti-single stranded DNA antibody (anti-ssDNAab) is used as a marker for systemic lupus erythematosus. We found a 'prozone-like phenomenon,' which was different from an original prozone phenomenon, in chemiluminescent enzyme immunoassay using magnetic particles for the measurement of serum anti-ssDNAab titers. We investigated mechanisms of the prozone-like phenomenon and countermeasures to prevent it from being overlooked. METHODS: This study examined 679 samples from patients tested for anti-ssDNAab titer at our hospital. In addition, the BF photometry OD value 2 (OD2), an index of optical density, was monitored simultaneously. RESULTS: The undiluted samples with a prozone-like phenomenon showed extremely lower OD2. Those samples were able to be distinguished from other samples by setting OD2 criteria based on the 95% prediction interval. Significant differences (p < 0.05) were found in the titer ratios (ten-fold diluted against undiluted) between groups with >1.5 and other groups with <1.5 for the ratios of OD2. CONCLUSIONS: We proposed two valuable methods to find a prozone-like phenomenon: one by setting OD2 criteria based on the 95% prediction interval and the other by analyzing the ratios both in titers and OD2 between undiluted and 10-fold diluted samples.

Continuous biomarker monitoring by particle mobility sensing with single molecule resolution.

Healthcare is in demand of technologies for real-time sensing in order to continuously guard the state of patients. Here we present biomarker-monitoring based on the sensing of particle mobility, a concept wherein particles are coupled to a substrate via a flexible molecular tether, with both the particles and substrate provided with affinity molecules for effectuating specific and reversible interactions. Single-molecular binding and unbinding events modulate the Brownian particle motion and the state changes are recorded using optical scattering microscopy. The technology is demonstrated with DNA and protein as model biomarkers, in buffer and in blood plasma, showing sensitivity to picomolar and nanomolar concentrations. The sensing principle is direct and self-contained, without consuming or producing any reactants. With its basis in reversible interactions and single-molecule resolution, we envisage that the presented technology will enable biosensors for continuous biomarker monitoring with high sensitivity, specificity, and accuracy.

Reduced rate of DNA replication fork movement in megaloblastic anemia.

Chromatography on benzoylated naphthoylated DEAE-cellulose has been used to fractionate fully double-stranded from partially single-stranded DNA molecules. DNA was extracted from phytohemagglutinin-stimulated lymphocytes from patients with megaloblastic anemia resulting from vitamin B12 or folate deficiency after pulse-labeling the cells with [3H]thymidine for 5 min and chasing in unlabeled medium for 24 h. No gross accumulation of partially single-stranded material was observed in the DNA of these cells when compared with DNA from similarly labeled control cells obtained by the addition of 5-formyl tetrahydrofolic acid to the culture medium. When DNA from lymphocytes labeled with a 5-min pulse of [3H]thymidine and sheared to fragments of an average length of 18 micrometer was chromatographed on benzoylated naphthoylated DEAE-cellulose, approximately 80% of the label was recovered in the partially single-stranded fraction. After chasing in unlabeled medium the label was progressively transferred to the double-stranded fraction over a period of 2--3 h. The rate of transfer was slower in megaloblastic lymphocytes than in controls. The difference in rate suggested a slower rate of replication fork movement in megaloblastic lymphocytes and so the density shift technique of Painter and schaeffer (J. Mol. Biol. 45: 467--479, 1969) was used to measure the fork rate directly. [3H]Deoxycytidine was used as the labeled nucleoside to avoid possible complications arising from [3H]thymidine labeling of megaloblastic cells. Investigations on the lymphocytes from four patients showed that the replication fork rate in vitamin-treated control lyphocytes was about 1 micrometer/min. The fork rates in the corresponding untreated cells were invariably lower and rates ranging from 40 to 92% of those of controls were observed. Normal lymphocytes treated with the deoxynucleotide pool-depleting drugs methotrexate or hydroxyurea displayed defects in DNA synthesis similar to those of untreated megaloblastic lymphocytes. We propose that the delayed DNA replication fork movement in cells of patients with megaloblastic anemia results from impaired biosynthesis of DNA precursors.

The occurrence of single-stranded DNA in the serum of patients with systemic lupus erythematosus and other diseases.

Single-stranded DNA (SDNA) occurs in high incidence and in greatest concentration in the sera of patients with systemic lupus erythematosus (SLE), where levels as high as 250 mug/ml were observed. SDNA appears to be an imunogen for anti-SDNA antibodies and forms complexes in vivo of both anti-SDNA-SDNA and anti-NDNA-SDNA types, which apparently play a role in the pathogenesis of the glomerulonephritis found in patients with SLE, SDNA is also found in high incidence but at lower levels in the sera of patients with rheumatoid arthritis. Lesser amounts of SDNA are found in several other diseases in which a low incidence of anti-SDNA antibodies is observed.