ABSTRACT
OBJECTIVES: The Epley maneuver (EM) shows immediate effect, wherein disappearance of positional nystagmus occurs soon after the EM. Our previous study showed that setting interval times during the EM reduced the immediate effect. The purpose of this study is to identify the head position for which interval time reduces the immediate effect. METHODS: Fifty-one patients with posterior canal type of benign paroxysmal positional vertigo (BPPV) were randomly assigned to the following three groups: 10 min interval time set at the first head position of the EM in group A, at the third head position in group B, and at the fourth head position in group C. The primary outcome measure (POï¼) was the ratio of maximum slow-phase eye velocity of positional nystagmus soon after the EM, compared with that measured before the EM. A large ratio value indicates a poor immediate effect of the EM. RESULTS: The POï¼ in group A (0.07) was smallest (B: 0.36, C: 0.49) (p < 0.001). DISCUSSION: The interval times at the third and fourth head positions reduced the immediate effect of the EM. Our previous study showed that the effect of BPPV fatigue is continued by maintaining the first head position of the EM. BPPV fatigue constitutes fatigability of positional nystagmus with repeated performance of the Dix-Hallpike test. Our findings may be interpreted in accordance with the theory that the immediate effect of the EM is BPPV fatigue itself, because we observed that the effect of BPPV fatigue is strongest in group A.
ABSTRACT
Objective: Hybrid of reversed image of positive endolymph signal and negative image of perilymph signal (HYDROPS) in delayed gadolinium-enhanced magnetic resonance imaging (MRI) typically depicts normal inner ear as "white-tone" and endolymphatic hydrops as "black-transparent" appearances, whereas ears with auditory and vestibular disorders are occasionally depicted as "gray-tone." This study aimed to investigate the pathological basis of sudden sensorineural hearing loss (SSNHL) patients with "gray-tone" appearances on HYDROPS. Methods: Delayed gadolinium-enhanced MRI examinations were conducted on 29 subjects with unilateral SSNHL. We mainly analyzed positive perilymph image (PPI) and positive endolymph image (PEI), which were components HYDROPS. Results: On PPI, signal intensity (SI) values extracted from the cochlear and vestibular region of interest (ROI) were higher in the SSNHL ears with dizziness/vertigo symptom at the first visit compared to the healthy ear. Additionally, the PPI/PEI enhancement pattern in the vestibule was associated with a high prevalence of hearing and vestibular deteriorations at the first visit and poor hearing improvement after treatment. Conclusion: Enhancement on PPI/PEI may result from leakage of gadolinium into the inner ear following breakdown of the blood-labyrinth barrier, with high SI being correlated with the amount of leakage. Particularly, a significant leakage into the endolymphatic space, defined as PPI+/PEI+, indicates severe inner ear pathology. Ultimately, we emphasize that the "gray-tone" appearance in the inner ear on HYDROPS comprises enhancements on both PPI and PEI and propose a new classification for evaluating SSNHL Peri- and Endolymphatic image Enhancement pattern in Delayed gadolinium-enhanced MRI (SPEED). Level of Evidence: 4.
ABSTRACT
Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/µL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.
Subject(s)
Biosensing Techniques , RNA , Biosensing Techniques/methods , Nucleic Acid Amplification Techniques/methods , DNA-Directed DNA PolymeraseABSTRACT
OBJECTIVES: Since the first report by Hallpike and Yamakawa in 1938, many more patients with Meniere's disease (MD) with endolymphatic hydrops (EHs) have been described. Mental/physical stress and a subsequent increase in the release of the anti-diuretic hormone (ADH) supposedly triggers MD. In the present study, to assess the relationship between stress and EHs, we conducted a series of stress-related questionnaires as well as a 3D endolymphatic space (ELS) analysis in patients with unilateral MD. METHODS: We enrolled 76 patients with unilateral MD (uMD) as the active group and 75 patients with unilateral benign paroxysmal positional vertigo (uBPPV) as the control group; both underwent examinations between June 2014 and November 2019. All patients underwent 3-T magnetic resonance imaging (MRI) 4 h after intravenous gadolinium injection. We used the total fluid space (TFS), ELS, and ELS rate (ELS/TFS × 100), which is the percentage of the volume of the ELS relative to that of the TFS, for a precise evaluation of the ELS and EHs in MD. Stress was evaluated using the Self-Rating Depression Scale (SDS), the psychological Stress Response Scale (SRS), and the modified Dizziness Handicap Inventory (mDHI). Stress scores and blood ADH levels were compared across patient groups. RESULTS: In patients with uMD, ELS rates significantly correlated with SRS scores on both the affected and the healthy side and with mDHI scores on the affected side, while the SDS and ADH showed no significant correlation with the ELS rates. Correlations were much stronger in the group with severe SDS and one with low ADH levels. CONCLUSIONS: The present results indicate that stress may be involved in EHs development in uMD, not only in the ipsilateral but also the contralateral ear. They also suggest that patients with neuropsychiatric tendencies may develop EHs and MD in response to a stressful lifestyle.
Subject(s)
Ear, Inner , Endolymphatic Hydrops , Meniere Disease , Humans , Meniere Disease/diagnostic imaging , Ear, Inner/diagnostic imaging , Endolymphatic Hydrops/diagnostic imaging , Benign Paroxysmal Positional Vertigo , Injections, Intravenous , Magnetic Resonance ImagingABSTRACT
OBJECTIVE: To elucidate the relationship between vertigo and EH volume after medical treatment, we investigated changes in endolymphatic hydrops (EH) volume using inner ear magnetic resonance imaging (ieMRI) in relation to clinical results for vertigo and hearing after administration of the anti-vertiginous medications betahistine, adenosine triphosphate (ATP), isosorbide (ISO), and saireito (SAI) for Meniere's disease (MD). METHODS: We retrospectively enrolled 202 consecutive patients diagnosed with unilateral MD from 2015 to 2021 and assigned them to four groups: Group I (G-I), symptomatic oral medication with betahistine only (CONT); Group II (G-II), inner ear vasoactive oral medication (ATP); Group III (G-III), osmotic diuretic oral medication (ISO); and Group IV (G-IV), kampo oral medication (SAI). In total, 172 patients completed the planned one-year-follow-up, which included the assessment of vertigo frequency, hearing improvement, and changes in EH using ieMRI (G-I, n=40; G-II, n=42; G-III, n=44; G-IV, n=46). We constructed 3D MRI images semi-automatically and fused the 3D images of the total fluid space (TFS) of the inner ear and endolymphatic space (ELS). After fusing the images, we calculated the volume ratios of the TFS and ELS (ELS ratios). RESULTS: One year after treatment, vertigo was controlled with zero episodes per month in 57.5% (23/40) of patients in G-I, 78.6% (33/42) in G-II, 81.8% (36/44) in G-III, and 82.6% (38/46) in G-IV (statistical significance: G-I
Subject(s)
Endolymphatic Hydrops , Meniere Disease , Vestibule, Labyrinth , Humans , Meniere Disease/diagnostic imaging , Meniere Disease/drug therapy , Meniere Disease/pathology , Retrospective Studies , Betahistine/therapeutic use , Endolymphatic Hydrops/diagnostic imaging , Endolymphatic Hydrops/drug therapy , Vertigo/diagnostic imaging , Vertigo/drug therapy , Magnetic Resonance Imaging/methodsABSTRACT
OBJECTIVE: The caloric test (C-test) and video head impulse test (vHIT) are known to occasionally show contradictory results in patients with Meniere's disease (MD). The reasons underlying this discrepancy between the two tests are currently unclear. We aimed to reveal the mechanisms responsible for this discrepancy by performing volumetric evaluation of the endolymphatic space (ELS) by using endoluminal contrast-enhanced inner ear MRI (ieMRI). METHODS: We enrolled 136 patients (174 ears) who visited the vertigo/dizziness center of our university and underwent the C-test and vHIT between February 2018 and February 2020. Inner ear MRI was also performed to determine the presence of endolymphatic hydrops (EH). The percentage of patients diagnosed with each vestibular disease was as follows: MD, 23.0%; benign paroxysmal positional vertigo (BPPV), 17.8%; bilateral vestibular disorder (BVD), 9.2%; sudden deafness with vertigo (SD), 8.0%; peripheral dizziness (PD), 7.5%; unilateral vestibular disorder (UVD), 6.9%; vestibular neuritis (VN), 6.3%; delayed endolymphatic hydrops (DEH), 3.4%; central dizziness (CD), 2.9%; Hunt syndrome (Hunt), 1.2%; and other disorders (OD), 13.8%. RESULTS: Among the ears in the present study, 46.0% (80/174) showed a discrepancy in the results of the C-test and vHIT, and the disease-related distribution of patients showing this discrepancy was as follows; MD, 38.8% (27/80; p = 0.0019); BVD, 13.8% (11/80); UVD, 12.5% (10/80); SD, 7.5% (6/80); BPPV, 6.3% (5/80); PD, 6.3% (5/80); VN, 3.8% (3/80); DEH, 3.8% (3/80); CD, 2.5% (2/80); Hunt, 0.0% (0/80); and OD, 10.0% (8/80). In all cases, the discrepancy presented as a positive C-test result and negative vHIT result. The ELS ratio was measured for the whole inner ear, cochlea, vestibule, and semicircular canal, and the relationships between the rates and the presence of discrepancy was examined. Inner ear ELS ratio was 17.9% ± 10.8% in patients with the discrepancy and 15.2% ± 8.8% in those without the discrepancy (p = 0.036). Cochlear ELS ratio was 14.9% ± 11.3% in patients with the discrepancy and 11.9% ± 10.3% in those without the discrepancy (p = 0.0012). Vestibular ELS ratio was 22.3% ± 16.2% in patients with the discrepancy and 17.2% ± 12.7% in those without the discrepancy (p = 0.032). Semicircular canal ELS ratio was 18.0% ± 11.0% in patients with the discrepancy and 16.5% ± 9.6% in those without the discrepancy (p = 0.442). CONCLUSION: The volume of the ELS may affect the discrepancy of results between the C-test and vHIT.
Subject(s)
Endolymphatic Hydrops , Meniere Disease , Vestibular Neuronitis , Vestibule, Labyrinth , Humans , Caloric Tests/methods , Dizziness , Head Impulse Test/methods , Meniere Disease/diagnostic imaging , Vestibule, Labyrinth/diagnostic imaging , Vestibular Neuronitis/diagnosis , Benign Paroxysmal Positional Vertigo , Endolymphatic Hydrops/diagnostic imaging , Magnetic Resonance Imaging/methodsABSTRACT
OBJECTIVES: This study aimed to investigate the endolymphatic hydrops (EH)-positivity rates among patients with recurrent audiovestibular symptoms using intravenous injection of gadolinium-enhanced inner ear magnetic resonance imaging (ieMRI). METHODS: We reviewed 710 successive patients with recurrent audiovestibular symptoms at the Vertigo/Dizziness Center of Nara Medical University and other related hospitals, between May 2014 and April 2020. We performed ieMRI on 153 patients with unilateral recurrent cochleovestibular symptoms (rCV), 51 with recurrent vertigo symptoms (rVO), and 84 with unilateral recurrent cochlear symptoms (rCO). RESULTS: EH was observed in 69.4% of the participants: 81.7% in the rCV group, 19.6% in the rVO group, and 77.4% in the rCO group. The participants were divided into two groups according to the disease duration: short-duration and long-duration groups. In the short-duration group (less than 4 years), EH was observed in 82.3%, 42.9%, and 71.4% of the patients in rCV, rVO, and rCO groups, respectively; in the long-duration group (more than 5 years), EH was observed in 81.1%, 10.8%, and 81.6% of the patients in rCV, rVO, and rCO groups, respectively. CONCLUSIONS: The longer the duration of the disease, the larger the EH-positivity rates in patients with rCO, smaller in those with rVO, and unchanged in those with rCV. Although ieMRI could not detect EH with 100% accuracy in Ménière's disease, the present pathological statistics of patients with recurrent audiovestibular symptoms might be helpful in considering the pathology-based treatment strategy.
Subject(s)
Ear, Inner , Endolymphatic Hydrops , Meniere Disease , Ear, Inner/diagnostic imaging , Endolymphatic Hydrops/diagnostic imaging , Gadolinium , Humans , Magnetic Resonance Imaging/methods , Meniere Disease/diagnostic imagingABSTRACT
Meniere's disease is a common inner ear disorder accompanied by vertigo attacks and fluctuating hearing loss that some believe is due to a stressful lifestyle. To elucidate the scientific relationship in neuro-endocrinology between Meniere's disease and stress, we examined the surgical results of endolymphatic sac drainage surgery and changes in stress-induced plasma arginine-vasopressin levels. We enrolled 100 intractable Meniere's patients and examined surgical results and plasma vasopressin levels. Fifty-four chronic otitis media patients who underwent tympano-mastoidectomy formed a control group. We assessed surgical results during a 2-year follow-up period, including vertigo and hearing loss. We examined plasma vasopressin levels just before surgery and 1 week, 1 year, and 2 years after surgery. In patients with intractable Meniere's disease, plasma vasopressin levels were significantly reduced 1 week after surgery compared to the decrease observed in chronic otitis media patients after tympano-mastoidectomy. In intractable Meniere's disease, long-lasting low plasma vasopressin levels after surgery were associated with significantly good surgical results. In recurrent Meniere's disease, a gradual plasma vasopressin level elevation was observed after surgery, followed by recurrent vertigo attacks and sensorineural hearing loss. It is suggested that long-lasting high levels of plasma vasopressin could have adverse effects on inner ear water metabolism and the subsequent Meniere's disease symptoms. Effective treatments for Meniere's disease might be best based on the maintenance of low plasma vasopressin levels.
ABSTRACT
We previously synthesized thioflavin T (ThT) with a hydroxyethyl group introduced at the N3-position (ThT-HE), which binds predominantly to the parallel G-quadruplex (G4) structure found in c-Myc and emits strong fluorescence. In this study, to investigate the effects of introduced substituents on G4 binding and fluorescence emission, a ThT derivative in which the hydroxyl group of ThT-HE was replaced with an amino group (ThT-AE) was synthesized for the first time. Furthermore, three other N3-modified ThT derivatives (ThT-OE2, ThT-SP, and ThT-OE11) having different substituent structures were synthesized by the N-acylation of the terminal amino group of ThT-AE, and their G4-binding and emission properties were investigated. The results showed that, although ThT-AE shows binding selectivity depending on the type of G4, its emission intensity is significantly decreased as compared to that of ThT-HE. However, ThT-OE11, which features an 11-unit oxyethylene chain attached to the terminal amino group of ThT-AE, regained about one-half of the emission intensity of ThT-HE while retaining selectivity for G4s. Accordingly, ThT-OE11 may be used as a key intermediate for synthesizing the conjugates of G4 binders and probes.
Subject(s)
Benzothiazoles/chemistry , Benzothiazoles/metabolism , G-Quadruplexes , Nitrogen/chemistry , Base Sequence , Genes, myc/genetics , Spectrometry, FluorescenceABSTRACT
Background: The caloric test has been used to evaluate the semi-circular canal function for decades. In 2009, the video head impulse test (vHIT) was introduced, which can be used to evaluate the semi-circular canal function within a short time. Although both tests examine the semi-circular canal, the stimulation methods differ and it is unclear whether the vHIT is equivocal to the caloric test.Aims/objectives: This study aimed to discern the differences between the vHIT and caloric test.Material and methods: This study comprised 112 patients with vertigo who visited the vertigo/dizziness centre at our university hospital. Each of these patients underwent a caloric test and vHIT within the same day, and their results were compared. Additionally, an electrocochleography (EcoG) examination, glycerol test (G test), and MRI (performed 4 h after an intravenous gadolinium injection) were conducted to evaluate the influence of endolymphatic hydrops (EH) on the caloric test and vHIT results.Results: Differences in the caloric test and vHIT results, among those with and without EH, were observed in 66.7 and 35.3% of patients, respectively.Conclusions and significance: EH resulted in a difference in results between the caloric test and vHIT. Activated hair cell type may also be implicated.
Subject(s)
Caloric Tests , Head Impulse Test , Meniere Disease/diagnosis , Semicircular Canals/physiopathology , Vestibular Diseases/diagnosis , Adult , Aged , Aged, 80 and over , Audiometry, Evoked Response , Female , Humans , Magnetic Resonance Imaging , Male , Meniere Disease/physiopathology , Middle Aged , Semicircular Canals/diagnostic imaging , Vertigo/etiology , Vertigo/physiopathology , Vestibular Diseases/physiopathologyABSTRACT
Human C-reactive protein (CRP) and lactate dehydrogenase are important markers in clinical laboratory testing-the former is used to detect in vivo inflammation, and the latter is used to detect cell necrosis and tissue destruction. We developed aptamers that bind to human CRP and human lactate dehydrogenase-5 (LDH-5) with high affinities (dissociation constants of 6.2 pM and 235 pM, respectively), applying the systematic evolution of ligands by exponential enrichment (SELEX) method, and by using a modified DNA library containing the following base-appended base modifications: analog adenine derivative at the fifth position of uracil (Uad), analog guanine derivative at the fifth position of uracil (Ugu), and analog adenine derivative at the seventh position of adenine (Aad). A potential application of these aptamers as sensor elements includes high-sensitivity target detection in point-of-care testing.
Subject(s)
Aptamers, Nucleotide/genetics , C-Reactive Protein/genetics , Lactate Dehydrogenase 5/genetics , Aptamers, Nucleotide/chemistry , Base Sequence , DNA, Single-Stranded , Gene Library , Humans , Molecular Structure , SELEX Aptamer TechniqueABSTRACT
We show herein the highly effective inhibition of tumor cell growth using a gel consisting of a fibrin polymer formed with the in situ condensation of a camptothecin (CPT) derivative as an anti-cancer drug, which is efficiently conveyed with a carrier aptamer from a solution to the gel in a phenomenon, called selective oligonucleotide entrapment in fibrin polymers (SOEF).
Subject(s)
Antineoplastic Agents/chemistry , Fibrin/chemistry , Antineoplastic Agents/pharmacology , Aptamers, Nucleotide/chemistry , Camptothecin/chemistry , Camptothecin/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Gels/chemistry , Humans , Microscopy, Fluorescence , Polymers/chemistryABSTRACT
Background: Endolymphatic sac drainage (ELSD) may have a positive effect on endolymphatic hydrops (EH) and may help to preserve inner ear function. However, the relationship between changes in EH volumes and hearing function after ELSD has not been described. Objectives: We aimed to reveal the factors related to changes in hearing and EH following ELSD. Material and Methods: Twenty-one patients who received ELSD were enrolled. Pure tone audiometry and 3-T magnetic resonance imaging (MRI) 4 h after intravenous injection of gadolinium enhancement were performed just before surgery and 2 years later. To characterize the endolymphatic space (ELS), we measured the volume of the total fluid (TFS) and ELS and calculated the ratio of ELS to TFS (ELS ratio). Results: The ELS ratio of the patients who showed hearing improvement was 18.5 ± 11.4% before surgery and 23.9 ± 14.3% after. For those with no change, it was 29.7 ± 10.8% before and 29.4 ± 9.5% after, and in patients with worsened hearing function it was 22.7 ± 7.5% before and 27.2 ± 13.4% after. Conclusion: We found no correlation between the changes in hearing function and the volume of EH after ELSD.
Subject(s)
Drainage/methods , Endolymphatic Hydrops/diagnosis , Endolymphatic Hydrops/therapy , Imaging, Three-Dimensional , Magnetic Resonance Imaging/methods , Meniere Disease/therapy , Adult , Analysis of Variance , Audiometry, Evoked Response , Audiometry, Pure-Tone , Cohort Studies , Contrast Media , Endolymphatic Sac/diagnostic imaging , Endolymphatic Sac/pathology , Female , Gadolinium , Humans , Male , Meniere Disease/diagnosis , Middle Aged , Prognosis , Recovery of Function , Retrospective Studies , Risk Assessment , Severity of Illness Index , Treatment OutcomeABSTRACT
Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, for these methods, improved rapidity and simplicity have been required for relevant techniques in laboratory research and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N3-( d-desthiobiotinyl-PEGylated) thioflavin T (ThT-PD) and N3-(cortisolyl-PEGylated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. Compared to those of the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with the target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N3-ThT derivatives by the rapid specific staining of glucocorticoid receptors in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took â¼24 h for immunostaining to capture the corresponding fluorescence images.
Subject(s)
Benzothiazoles/chemistry , Fluorescent Dyes/chemistry , Molecular Imaging/methods , Receptors, Glucocorticoid/metabolism , Humans , MCF-7 Cells , Spectrometry, Fluorescence/methodsABSTRACT
We found for the first time that a thrombin-binding DNA aptamer (TBA) is selectively entrapped in fibrin gels during the gel growth reaction catalyzed by thrombin. Furthermore, using this phenomenon, we successfully demonstrated multiple incorporation of amphiphilic aliphatic groups into fibrin gels via chemically modified TBA.
Subject(s)
Aptamers, Nucleotide/chemistry , Fibrin/chemistry , Aptamers, Nucleotide/metabolism , Base Sequence , Cell Proliferation/drug effects , Fibrin/metabolism , Fibrin/pharmacology , Fibrinogen/metabolism , Gels/chemistry , HeLa Cells , Humans , Microscopy, Fluorescence , Thrombin/metabolismABSTRACT
Gene regulation systems are mimicked by simple quantitative detection of non-nucleic acid molecular targets such as protein and metabolite. Here, we describe a one-tube, one-step real-time quantitative detection methodology for isothermal signal amplification of those targets. Using this system, real-time quantitative detection of thrombin and streptomycin, which were used as examples for protein and metabolite targets, was successfully demonstrated with detection limits of at most 50 pM and 75 nM, respectively. Notably, the dynamic range of target concentrations could be obtained for over four orders of magnitude. Thus, our method is expected to serve as a point-of-care or on-site test for medical diagnosis and food and environmental hygiene.
Subject(s)
Biosensing Techniques/methods , Molecular Diagnostic Techniques/methods , Point-of-Care Systems , Streptomycin/analysis , Thrombin/analysis , Transcription, GeneticABSTRACT
We have attained a chemically modified DNA aptamer against salivary α-amylase (sAA), which attracts researchers' attention as a useful biomarker for assessing human psychobiological and social behavioural processes, although high affinity aptamers have not been isolated from a random natural DNA library to date. For the selection, we used the base-appended base (BAB) modification, that is, a modified-base DNA library containing (E)-5-(2-(N-(2-(N6-adeninyl)ethyl))carbamylvinyl)-uracil in place of thymine. After eight rounds of selection, a 75 mer aptamer, AMYm1, which binds to sAA with extremely high affinity (Kd < 1 nM), was isolated. Furthermore, we have successfully determined the 36-mer minimum fragment, AMYm1-3, which retains target binding activity comparable to the full-length AMYm1, by surface plasmon resonance assays. Nuclear magnetic resonance spectral analysis indicated that the minimum fragment forms a specific stable conformation, whereas the predicted secondary structures were suggested to be disordered forms. Thus, DNA libraries with BAB-modifications can achieve more diverse conformations for fitness to various targets compared with natural DNA libraries, which is an important advantage for aptamer development. Furthermore, using AMYm1, a capillary gel electrophoresis assay and lateral flow assay with human saliva were conducted, and its feasibility was demonstrated.
Subject(s)
Aptamers, Nucleotide/chemistry , Saliva/chemistry , Biomarkers/analysis , Humans , SELEX Aptamer Technique/methods , Uracil/analogs & derivativesABSTRACT
Nucleic acid aptamers for small molecules are currently being developed and have a potential role in diverse applications including biosensing, diagnostics, and therapeutics involving low-molecular-weight biomarkers and drugs. To enhance and broaden their functions through chemical modification, systematic evolution of ligands by exponential enrichment (SELEX) selection has been attempted with modified DNA/RNA libraries. Recently, we demonstrated the superior efficacy of base modification for affinity enhancement and the usefulness of unnatural nucleic acid libraries for development of small-molecule aptamers. In this unit, we describe construction of a modified DNA library that includes (E)-5-(2-(N-(2-(N(6) -adeninyl)ethyl))carbamylvinyl)uracil bases and acquisition of high-affinity camptothecin-binding DNA aptamers, in addition to those of the corresponding natural DNA library and aptamers, using the SELEX method. © 2016 by John Wiley & Sons, Inc.
Subject(s)
Aptamers, Nucleotide/chemistry , Camptothecin/chemistry , Small Molecule Libraries/chemistry , Evolution, Molecular , Gene Library , Ligands , Uracil/analogs & derivatives , Uracil/chemistryABSTRACT
We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.
Subject(s)
Biomarkers/analysis , DNA, Circular/genetics , Nucleic Acid Amplification Techniques/methods , RNA/genetics , G-Quadruplexes , Limit of DetectionABSTRACT
Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.