scholarly journals Harnessing intrinsic fluorescence for typing of secondary structures of DNA

2020 ◽  
Author(s):  
Michela Zuffo ◽  
Aurélie Gandolfini ◽  
Brahim Heddi ◽  
Anton Granzhan
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
Emission Spectra ◽  
Principal Component ◽  
Secondary Structures ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Linear Discriminant ◽  
Effective Manner

ABSTRACTDNA is polymorphic since, despite its ubiquitous presence as a double-stranded helix, it is able to fold into a plethora of other secondary structures both in vitro and in cells. Despite the considerable advances that have been made in understanding this structural diversity, its high-throughput investigation still faces severe limitations. This mainly stems from the lack of suitable label-free methods, combining a fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of the suitability of this phenomenon for tracking the conformational changes of DNA, we examined the intrinsic steady-state emission spectra of an 89-membered set of synthetic oligonucleotides with reported conformation (G-quadruplexes, i-motifs, single- and double stranded DNA) by means of multivariate analysis. Specifically, principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, albeit without discrimination between single- and double-stranded structures. Linear discriminant analysis of the same training set was exploited for the assessment of new sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labelling agent or dye, avoiding the related intrinsic bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T), the most fluorescent G4 structure reported to date. This property is likely to arise from the similar base-stacking geometry in both types of structures.

scholarly journals Harnessing intrinsic fluorescence for typing of secondary structures of DNA

2020 ◽  
Vol 48 (11) ◽  
pp. e61-e61 ◽  
Author(s):  
Michela Zuffo ◽  
Aurélie Gandolfini ◽  
Brahim Heddi ◽  
Anton Granzhan
Keyword(s):  
Nucleic Acids ◽  
High Throughput ◽  
Conformational Changes ◽  
Emission Spectra ◽  
Principal Component ◽  
Structural Diversity ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Linear Discriminant ◽  
Effective Manner

Abstract High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).

scholarly journals Activation of GCN2 by the ribosomal P-stalk

2019 ◽  
Vol 116 (11) ◽  
pp. 4946-4954 ◽  
Author(s):  
Alison J. Inglis ◽  
Glenn R. Masson ◽  
Sichen Shao ◽  
Olga Perisic ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

scholarly journals Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

2020 ◽  
Vol 48 (20) ◽  
pp. e120-e120 ◽  
Author(s):  
Obtin Alkhamis ◽  
Weijuan Yang ◽  
Rifat Farhana ◽  
Haixiang Yu ◽  
Yi Xiao
Keyword(s):  
Ligand Binding ◽  
Small Molecule ◽  
High Throughput ◽  
Dna Aptamer ◽  
Label Free ◽  
General Applicability ◽  
Gold Standard Method ◽  
Characterization Methods ◽  
Increased Risk

Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.

scholarly journals Effects of Xylo-Oligosaccharide on the Gut Microbiota of Patients With Ulcerative Colitis in Clinical Remission

2021 ◽  
Vol 8 ◽  
Author(s):  
Zongwei Li ◽  
Zhengpeng Li ◽  
Liying Zhu ◽  
Ning Dai ◽  
Gang Sun ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gut Microbiota ◽  
Healthy Volunteers ◽  
Clinical Remission ◽  
Principal Component ◽  
Fecal Samples ◽  
Linear Discriminant ◽  
Wilcoxon Rank Sum Test ◽  
Rdna Sequencing

Gut microbiota dysbiosis is closely associated with ulcerative colitis (UC). Prebiotic therapy is a potential approach for UC management especially remission maintaining. Xylo-oligosaccharide (XOS) is an efficient prebiotic with proven health benefits and few side effects. However, the effects of XOS on the gut microbiota of patients with UC have not been investigated previously. The aim of this study was to evaluate the prebiotic effects of XOS on the fecal microbiota of patients with UC in clinical remission using an in vitro fermentation model. Five patients with UC in clinical remission and five healthy volunteers were enrolled in this study. Fresh fecal samples of UC patients were diluted and inoculated in yeast extract, casitone and fatty acid (YCFA) medium alone or with XOS. After fermentation for 48 h, samples were collected for 16S rDNA sequencing to investigate the gut microbiota composition. Differences in the gut microbiota between healthy volunteers and UC patients in clinical remission were detected using original fecal samples. Subsequently, the differences between the YCFA medium alone or with XOS samples were analyzed to illustrate the effects of XOS on the gut microbiota of UC patients. In both principal coordinate analysis (PCoA) and principal component analysis (PCA), the fecal samples of UC patients differed from those of healthy volunteers. Linear discriminant analysis effect size (LEfSe) analysis revealed that the relative abundances of g_Roseburia and g_Lachnospiraceae_ND3007_group were higher in healthy volunteers than in UC patients, while o_Lactobacillales abundance showed the opposite trend (P < 0.05). Wilcoxon rank-sum test bar plot showed that the abundances of g_Eubacterium_halli_group and g_Lachnospiraceae_ND3007_group were higher in the healthy volunteers than in the UC patients (P < 0.05). In addition, in UC patients, the Wilcoxon rank-sum test showed that XOS fermentation promoted the growth of bacterial groups including g_Roseburia, g_Bifidobacterium, and g_Lactobacillus, which is beneficial for recovery of intestinal diseases. These results suggest that XOS can relieve dysbiosis in the feces of UC patients in clinical remission and thus represent a potential prebiotic material for maintaining remission.

scholarly journals Systematic Identification of Antiprion Drugs by High-Throughput Screening Based on Scanning for Intensely Fluorescent Targets

2005 ◽  
Vol 79 (12) ◽  
pp. 7785-7791 ◽  
Author(s):  
Uwe Bertsch ◽  
Konstanze F. Winklhofer ◽  
Thomas Hirschberger ◽  
Jan Bieschke ◽  
Petra Weber ◽  
...  
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Prion Diseases ◽  
Inhibitory Effect ◽  
Helical Conformation ◽  
Response Curves ◽  
Vitro Assay ◽  
Systematic Identification

ABSTRACT Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrPSc), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrPC/PrPSc interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrPSc propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrPC and PrPSc is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

scholarly journals FTIR Microspectroscopy Coupled with Two-Class Discrimination Segregates Markers Responsible for Inter- and Intra-Category Variance in Exfoliative Cervical Cytology

2008 ◽  
Vol 3 ◽  
pp. BMI.S592 ◽  
Author(s):  
Michael J. Walsh ◽  
Maneesh N. Singh ◽  
Helen F. Stringfellow ◽  
Hubert M. Pollock ◽  
Azzedine Hammiche ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Cell Types ◽  
Cervical Cytology ◽  
Low Grade ◽  
High Grade ◽  
Linear Discriminant ◽  
Ftir Microspectroscopy ◽  
Category Variance ◽  
Different Cell Types

Infrared (IR) absorbance of cellular biomolecules generates a vibrational spectrum, which can be exploited as a “biochemical fingerprint” of a particular cell type. Biomolecules absorb in the mid-IR (2–20 μm) and Fourier-transform infrared (FTIR) microspectroscopy applied to discriminate different cell types (exfoliative cervical cytology collected into buffered fixative solution) was evaluated. This consisted of cervical cytology free of atypia (i.e. normal; n = 60), specimens categorised as containing low-grade changes (i.e. CIN1 or LSIL; n = 60) and a further cohort designated as high-grade (CIN2/3 or HSIL; n = 60). IR spectral analysis was coupled with principal component analysis (PCA), with or without subsequent linear discriminant analysis (LDA), to determine if normal versus low-grade versus high-grade exfoliative cytology could be segregated. With increasing severity of atypia, decreases in absorbance intensity were observable throughout the 1,500 cm–1 to 1,100 cm–1 spectral region; this included proteins (1,460 cm–1), glycoproteins (1,380 cm–1), amide III (1,260 cm–1), asymmetric (νas) PO2– (1,225 cm–1) and carbohydrates (1,155 cm–1). In contrast, symmetric (νs) PO2–(1,080 cm–1) appeared to have an elevated intensity in high-grade cytology. Inter-category variance was associated with protein and DNA conformational changes whereas glycogen status strongly influenced intra-category. Multivariate data reduction of IR spectra using PCA with LDA maximises inter-category variance whilst reducing the influence of intra-class variation towards an objective approach to class cervical cytology based on a biochemical profile.

scholarly journals RIGHT-ANGLE FLUORESCENCE SPECTROSCOPY FOR DIFFERENTIATION OF DISTILLED ALCOHOLIC BEVERAGES

2013 ◽  
Vol 12 (2) ◽  
pp. 83-92 ◽  
Author(s):  
Veronika Uríčková ◽  
Jana Sádecká ◽  
Pavel Májek
Keyword(s):  
Fluorescence Spectroscopy ◽  
Emission Spectra ◽  
Principal Component ◽  
Hierarchical Cluster ◽  
Alcoholic Beverages ◽  
Front Face ◽  
Excitation Wavelength ◽  
Spectroscopic Techniques ◽  
Excitation Spectra ◽  
Linear Discriminant

Abstract Total luminescence and synchronous scanning fluorescence spectroscopic techniques were investigated for differentiating brandies from mixed wine spirits. The studies were performed on 16 brandies from 3 different producers and 30 mixed wine spirits from 5 different producers. Differentiation between samples was accomplished by multivariate data analysis methods (principal component analysis, hierarchical cluster analysis, and linear discriminant analysis). Correct classification was obtained using emission spectra (400-650 nm) recorded at excitation wavelength 390 nm, excitation spectra (225-460 nm) obtained at emission wavelength 470 nm and synchronous fluorescence spectra (200-700 nm) collected at wavelength interval 80 nm. These results indicate that right-angle fluorescence spectroscopy offers a promising approach for the authentication of brandies as neither sample preparation nor special qualification of the personnel are required, and data acquisition and analysis are relatively simple when compared to front-face technique.

scholarly journals Ultrafast and Predictive Mass Spectrometry–Based Autotaxin Assays for Label-Free Potency Screening

2017 ◽  
Vol 22 (4) ◽  
pp. 425-432 ◽  
Author(s):  
Tom Bretschneider ◽  
Andreas Harald Luippold ◽  
Helmut Romig ◽  
Daniel Bischoff ◽  
Klaus Klinder ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Whole Blood ◽  
Label Free ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Data Points ◽  
Set Up

Autotaxin (ATX) is a promising drug target for the treatment of several diseases, such as cancer and fibrosis. ATX hydrolyzes lysophosphatidyl choline (LPC) into bioactive lysophosphatidic acid (LPA). The potency of ATX inhibitors can be readily determined by using fluorescence-based LPC derivatives. While such assays are ultra-high throughput, they are prone to false positives compared to assays based on natural LPC. Here we report the development of ultrafast mass spectrometry–based ATX assays enabling the measurement of data points within 13 s, which is 10 times faster than classic liquid chromatography–mass spectrometry. To this end, we set up a novel in vitro and whole-blood assay. We demonstrate that the potencies determined with these assays are in good agreement with the in vivo efficacy and that the whole-blood assay has the best predictive power. This high-throughput label-free approach paired with the translatable data quality is highly attractive for appropriate guidance of medicinal chemists for constructing strong structure-activity relationships.

Intrinsic fluorescence–based label-free detection of bovine amyloid A amyloidosis

2021 ◽  
pp. 104063872110492
Author(s):  
Naoki Ujike ◽  
Susumu Iwaide ◽  
Yuki Ono ◽  
Takayuki Okano ◽  
Tomoaki Murakami
Keyword(s):  
Analytical Data ◽  
Principal Component ◽  
Histochemical Staining ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Simple Method ◽  
Spectrometry Analysis ◽  
Amyloid A ◽  
Label Free Detection ◽  
Tissue Homogenates

Amyloidosis is diagnosed by the histologic detection of amyloid deposits; however, this method has limitations such as a prolonged diagnosis time and the need for histologic proficiency. We aimed to develop a rapid and simple method for diagnosing amyloidosis by targeting amyloid-specific endogenous fluorescence, which has not been reported previously, to our knowledge. Fluorescence fingerprint analysis of amyloid extracts and tissue homogenates derived from amyloid A (AA) amyloidosis-affected cattle exhibited a specific intrinsic fluorescence pattern. Furthermore, principal component analysis using analytical data revealed that AA could be identified by peaks near λex 350 nm and λem 430 nm. Fluorescence spectrometry analysis using tissue homogenates, which does not require special histochemical staining, enables the rapid detection of bovine AA.

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