Single-Molecule Analysis of i-motif Within Self-Assembled DNA Duplexes and Nanocircles

The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif–forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif–forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes

Single-Step FRET-Based Detection of Femtomoles DNA

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Multiplexed nucleic acid sensing with single molecule FRET

In recent years, biomarkers have drawn tremendous interest in the fields of clinical diagnostics and biotechnology. In fact, recent quests for biomarker analyses have discovered a myriad of disease-related biomarkers spanning a wide variety of biomolecules such as proteins, nucleic acids, and small molecule ligands. While the detection of only one target at a time is the most common practice in biosensing today, it has been shown that the accuracy of diagnosis increases significantly by measuring the level of multiple biomarkers instead of just one due to the possible overlap of certain biomarkers in more than one disease. However, currently available ensemble multiplexed methods such as DNA microarrays and polymerase chain reactions (PCR) often suffer from complicated design/engineering, semi-quantitative analysis, or a high false positive rate. Single molecule techniques can be an asset, in this regard, by providing unique insights into the behavior and interactions of individual molecules. For example, single molecule fluorescence resonance energy transfer (smFRET) is able to quantitatively measure signals from individual molecules and is a sensitive means for detection of biomarkers by relying on the emission from one or more donor/acceptor FRET pairs. However, current methods require multiple excitation sources and complex data analysis algorithms. To address this knowledge gap, using prism-based total internal reflection fluorescence (pTIRF) microscopy, we developed a FRET-based multiplexed detection method for DNA sequences utilizing only a single donor/acceptor fluorophores pair. By tuning the minimum distance separation between a FRET pair through a careful design, we showed that multiple FRET values can be achieved depending on the distance set. We utilized interconvertible hairpin-based sensors (iHabSs) where detection was dependent on a toehold-mediated displacement of a probe strand by the target. This approach allowed for simultaneous detection of 3 different DNA sequences with a detection limit of ~200 pM while still using a single FRET pair. To expand the application of the method to detect microRNA biomarkers that are present at low picomolar to femtomolar concentrations in biological samples, we have re-imagined our sensor design that offers a direct binding of target to the sensor and hence does not need go through the competitive toehold displacement process. Further, we implemented and refined a program called MASH-FRET (a MATLAB-based multifunctional analysis software for handling smFRET data) and evaluated its capability of identifying seemingly overlapped FRET populations. The motivation was to expand the multiplexing capability of predominately used single-molecule FRET techniques, which utilize only one FRET pair. Through MASH-FRET-enabled bootstrap-based analysis (also called BOBA-FRET) of experimental and simulated data, we first demonstrated that the statistical confidence of poorly resolved FRET populations can be readily determined. Using simulated data sets, we then demonstrated that the program can easily identify FRET populations that are separated only by ~0.1 FRET level, indicating the possibility of up to ~9-fold multiplexing. Overall, we showed that our streamlined MASH-FRET platform has great promise to increase the multiplexing capability of smFRET techniques without the need for complicated experimental set ups such as multiple FRET pairs and/or multiple excitation sources

Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers

The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (≤10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers

Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research

Exploring terminology for puerperal sepsis and its symptoms in urban Karachi, Pakistan to improve communication, care-seeking, and illness recognition

Puerperal sepsis is an important cause of maternal morbidity and mortality in developing countries. Awareness of local terminology for its signs and symptoms may improve communication about this illness, what actions to take when symptoms appear, timely care seeking, and clinical outcomes. This formative research aimed to improve recognition and management of postpartum sepsis in Pakistan by eliciting local terms used for postpartum illnesses and symptoms. We conducted 32 in-depth interviews with recently delivered women, their relatives, traditional birth attendants, and health care providers to explore postpartum experiences. Terms for symptoms and illness are used interchangeably (i.e. bukhar, the Urdu word for fever), many variations exist for the same term, and gradations of severity for each term as not associated with different types of illnesses. The lack of a designated term for postpartum sepsis in Urdu delays care-seeking and proper diagnosis, particularly at the community level. Ideally, a common lexicon for symptoms and postpartum sepsis would be developed but this may not be feasible or appropriate given the nature of the Urdu language and local understandings of postpartum illness. These insights can inform how we approach educational campaigns, the development of clinical algorithms that focus on symptoms, and counselling protocols

Scattering studies with low-energy kaon-proton femtoscopy in proton-proton collisions at the LHC

The study of the strength and behavior of the antikaon-nucleon ($\bar{K}N$) interaction constitutes one of the key focuses of the strangeness sector in low-energy quantum chromodynamics (QCD). In this Letter a unique high-precision measurement of the strong interaction between kaons and protons, close and above the kinematic threshold, is presented. The femtoscopic measurements of the correlation function at low pair-frame relative momentum of ($K^+p⊕K^-\bar{p}$) and ($K^-p⊕K^+\bar{p}$) pairs measured in pp collisions at s=5, 7, and 13 TeV are reported. A structure observed around a relative momentum of 58 MeV/c in the measured correlation function of ($K^-p⊕K^+\bar{p}$) with a significance of 4.4σ constitutes the first experimental evidence for the opening of the ($\bar{K}^0n⊕K^0\bar{n}$) isospin breaking channel due to the mass difference between charged and neutral kaons. The measured correlation functions have been compared to Jülich and Kyoto models in addition to the Coulomb potential. The high-precision data at low relative momenta presented in this work prove femtoscopy to be a powerful complementary tool to scattering experiments and provide new constraints above the $\bar{K}N$ threshold for low-energy QCD chiral models

Real-time data processing in the ALICE High Level Trigger at the LHC

International audienceAt the Large Hadron Collider at CERN in Geneva, Switzerland, atomic nuclei are collided at ultra-relativistic energies. Many final-state particles are produced in each collision and their properties are measured by the ALICE detector. The detector signals induced by the produced particles are digitized leading to data rates that are in excess of 48 GB/s. The ALICE High Level Trigger (HLT) system pioneered the use of FPGA- and GPU-based algorithms to reconstruct charged-particle trajectories and reduce the data size in real time. The results of the reconstruction of the collision events, available online, are used for high level data quality and detector-performance monitoring and real-time time-dependent detector calibration. The online data compression techniques developed and used in the ALICE HLT have more than quadrupled the amount of data that can be stored for offline event processing

Investigations of Anisotropic Flow Using Multiparticle Azimuthal Correlations in pppp, p−Pbp-Pb, Xe-Xe, and Pb-Pb Collisions at the LHC

International audienceMeasurements of anisotropic flow coefficients (vn) and their cross-correlations using two- and multiparticle cumulant methods are reported in collisions of pp at s=13  TeV, p-Pb at a center-of-mass energy per nucleon pair sNN=5.02  TeV, Xe-Xe at sNN=5.44  TeV, and Pb-Pb at sNN=5.02  TeV recorded with the ALICE detector. The multiplicity dependence of vn is studied in a very wide range from 20 to 3000 particles produced in the midrapidity region |η|v3>v4 is found in pp and p-Pb collisions, similar to that seen in large collision systems, while a weak v2 multiplicity dependence is observed relative to nucleus-nucleus collisions in the same multiplicity range. Using a novel subevent method, v2 measured with four-particle cumulants is found to be compatible with that from six-particle cumulants in pp and p-Pb collisions. The magnitude of the correlation between vn2 and vm2, evaluated with the symmetric cumulants SC(m,n) is observed to be positive at all multiplicities for v2 and v4, while for v2 and v3 it is negative and changes sign for multiplicities below 100, which may indicate a different vn fluctuation pattern in this multiplicity range. The observed long-range multiparticle azimuthal correlations in high multiplicity pp and p-Pb collisions can neither be described by pythia 8 nor by impact-parameter-Glasma, music, and ultrarelativistic quantum molecular dynamics model calculations, and hence, provide new insights into the understanding of collective effects in small collision systems