SELEX Experiment Research Articles

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A.

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM.

Production of the doubly charmed baryons at the SELEX experiment – The double intrinsic charm approach

The high production rate and $\langle x_{F} \rangle > 0.33$ of the doubly charmed baryons measured by the SELEX experiment is not amenable to perturbative QCD analysis. In this paper we calculate the production of the doubly heavy baryons with the double intrinsic charm Fock states whose existence is rigorously predicted by QCD. The production rate and the longitudinal momentum distribution are both reproduced. We also show that the production rates of the doubly charmed baryons and double $J/\psi $ production observed by NA3 collaboration are comparable. Recent experimental results are reviewed. The production cross section of the doubly charmed baryons at a fixed-target experiment at the LHC is presented.

The transverse momentum dependence of charged kaon Bose–Einstein correlations in the SELEX experiment

We report the measurement of the one-dimensional charged kaon correlation functions using 600GeV/cΣ−, π− and 540GeV/cp beams from the SELEX (E781) experiment at the Fermilab Tevatron. K±K± correlation functions are studied for three transverse pair momentum, kT, ranges and parameterized by a Gaussian form. The emission source radii, R, and the correlation strength, λ, are extracted. The analysis shows a decrease of the source radii with increasing kaon transverse pair momentum for all beam types.

Aptamer Selection Technology and Recent Advances.

Over the last decade, aptamers have begun to find their way from basic research to diverse commercial applications. The development of diagnostics is even more widespread than clinical applications because aptamers do not have to be extensively modified to enhance their in vivo stability and pharmacokinetics in diagnostic assays. The increasing attention has propelled the technical progress of the in vitro selection technology (SELEX) to enhance the efficiency of developing aptamers for commercially interesting targets. This review highlights recent progress in the technical steps of a SELEX experiment with a focus on high-throughput next-generation sequencing and bioinformatics. Achievements have been made in the optimization of aptamer libraries, separation schemes, amplification of the selected libraries and the identification of aptamer sequences from enriched libraries.

Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe.

Breast cancer is one of the most commonly diagnosed cancers among females worldwide. Early detection of breast cancer is of vital importance to the reduction of the mortality rate. However, the lack of specific biomarkers that can effectively identify breast cancer cells limits the ability for early diagnosis of breast cancer. RNA-cleaving fluorogenic DNAzymes (RFDs), which can be produced through the systematic evolution of ligands by exponential enrichment (SELEX) process, are catalytic DNA molecules capable of generating a fluorescent signal when the appropriate target is bound. In this study, we carried out a SELEX experiment to select for RFDs that are active in the cell lysate of MDA-MB-231, a model breast cancer cell line. We obtained a RFD probe, named AAI2-5, that can detect MDA-MB-231 at a concentration of cell lysate proteins as low as 0.5 μg/mL (which is equivalent to ∼5000 cell/mL). AAI2-5 is capable of distinguishing MDA-MB-231 cells from normal cells as well as other types of tumor cells, including other subtypes of breast cancer cells. Moreover, AAI2-5 responded positively to more than 90% of malignant breast tumors. This report is the first study to explore the RFD system for the detection of cancer cells. The results suggest that RFD can be potentially applied for the diagnosis and treatment of breast cancer in the future.

Hadronic production ofΞccat a fixed-target experiment at the LHC

In the paper, we present a detailed discussion on the $\Xi_{cc}$ production at a fixed target experiment at the LHC (After@LHC). The doubly charmed baryon $\Xi_{cc}$ is produced via the channel, ${\rm Proton} + {\rm Proton}\to\Xi_{cc}+X$. In estimating its hadroproduction, we discuss three dominant subprocesses, e.g. $g+g\to \Xi_{cc} +\bar{c} +\bar{c}$, $g+c\to \Xi_{cc}+\bar{c}$ and $c+c\to \Xi_{cc}+g$. During the production, it shall first generate a binding diquark and then form the $\Xi_{cc}$ baryon by grabbing soft light-quarks or gluons. We observe that both the two diquark configurations $(cc)[^3S_1]_{\bf\bar 3}$ and $(cc)[^1S_0]_{\bf 6}$ can have sizable contributions to the $\Xi_{cc}$ production. Large number of $\Xi_{cc}$ events can be generated at the After@LHC, whose total production cross section is larger than that of the SELEX experiment by about thirty-five times. It may also possible to study the properties of $\Xi_{bc}$ at the After@LHC. More specifically, we shall have about $8.3 \times 10^6$ $\Xi_{cc}$ events/year and $1.8 \times 10^4$ $\Xi_{bc}$ events/year when its integrated luminosity approaches to $2$ fb$^{-1}$/year. Thus, in addition to SELEX and LHC, the After@LHC shall provide another useful platform for studying the baryon properties.

Correlation femtoscopy of Kaons in the SELEX experiment

Preliminary results from the correlation femtoscopy of identical kaons in the SELEX experiment (Fermilab E781) are presented. Kaons are selected in inclusive reactions of Σ−C(Cu) interactions at an initial energy of 610 GeV. Kaon pairs are studied in all possible charged states: Ks0Ks0, Ks0K+, Ks0K−, K+K+, K+K− and K−K−. The source sizes of the hadronization region of neutral and charged kaon pairs are measured.

A model for sequential evolution of ligands by exponential enrichment (SELEX) data

A Systematic Evolution of Ligands by EXponential enrichment (SELEX) experiment begins in round one with a random pool of oligonucleotides in equilibrium solution with a target. Over a few rounds, oligonucleotides having a high affinity for the target are selected. Data from a high throughput SELEX experiment consists of lists of thousands of oligonucleotides sampled after each round. Thus far, SELEX experiments have been very good at suggesting the highest affinity oligonucleotide, but modeling lower affinity recognition site variants has been difficult. Furthermore, an alignment step has always been used prior to analyzing SELEX data. We present a novel model, based on a biochemical parametrization of SELEX, which allows us to use data from all rounds to estimate the affinities of the oligonucleotides. Most notably, our model also aligns the oligonucleotides. We use our model to analyze a SELEX experiment containing double stranded DNA oligonucleotides and the transcription factor Bicoid as the target. Our SELEX model outperformed other published methods for predicting putative binding sites for Bicoid as indicated by the results of an in-vivo ChIP-chip experiment.

Measurement of ΛΛ spin correlations in the SELEX experiment

We study HBT interferometry of ΛΛ pairs produced in ∑− A interactions using a ΛΛ spin composition measurement with data taken by SELEX, which accumulated data during the 1996–97 fixed target run at Fermilab. While the spin composition of a ΛΛ pair in the range of four-momentum difference of a pair Q < 1.5 GeV was found to be consistent with the suppression of spin state one, for Q > 1.5 GeV it was found to be consistent with statistical spin mixture. For the system the spin one fraction was found to be consistent with the expectation for a statistical spin mixture. We also estimated the dimension of the ΛΛ production region.

Spin correlations of Λ hyperons in the SELEX experiment

Two-particle correlations of the pairs of identical Λ hyperons in Σ− A interactions were studied based on the statistical data obtained in the SELEX experiment performed using the Tevatron collider at FNAL in 1996–1997. The preliminary results from the investigations of spin correlations of ΛΛ pairs are presented, and the dimension of the region of ΛΛ generation is estimated. The results are in good agreement with the world data and the result obtained using correlation function analysis.

Correlations of antiprotons with small relative momentumin the SELEX Experiment

The main results of the data processing and physical analysis of the SELEX experiment and the preliminary data on correlations of antiprotons with small relative momentum are presented. Correlation femtoscopy is a powerful tool for studying the spatial-temporal characteristics of the emission region of identical particles. The correlation functions obtained in the SELEX and STAR experiments are compared.

A Sol–Gel-Based Microfluidics System Enhances the Efficiency of RNA Aptamer Selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.

Measuring the masses of the charged hadrons using a RICH as a precision velocity spectrometer

The Selex experiment measured several billion charged hadron tracks with a high precision magnetic momentum spectrometer and high precision RICH velocity spectrometer. We have analysed these data to simultaneously measure the masses of all the long lived charged hadrons and anti-hadrons from the π to the Ω using the same detector and technique. The statistical precision achievable with this data sample is more than adequate for 0.1% mass measurements. We have used these measurements to develop and understand the systematic effects in using a RICH as a precision velocity spectrometer with the goal of measuring 10 masses with precision ranging from 100 KeV for the lightest to 1000 KeV for the heaviest. This requires controlling the radius measurement of RICH rings to the ∼ 10 − 4 level. Progress in the mass measurements and the required RICH analysis techniques developed are discussed.

Strange hadron production in Σ-A interactions in the SELEX experiment

We report our observations on the resonance signals of masses of 1520, 1670, 1810 (1 820), and 2100 MeV c−2, produced inclusively in the reaction Σ− + C(Cu) → p + K− + X, and the signals of masses of 1020 and 1525 MeV c−2, produced inclusively in the reaction Σ− + C(Cu) → K+ + K− + X, with a 600-GeV c−1- momentum hyperon beam in the SELEX experiment at the Fermilab. The masses, widths, and branching ratios were measured. The resonance signal of mass of 1520 MeV c−2 correspond to the known Λ(1520) hyperon. The signals of masses of 1020 and 1525 MeV c−2 in the K+K− system correspond to the φ0(1020)- and f2′(1525) mesons, respectively. The branching ratio of the resonance of mass of 1670 MeV c−2 relative to Λ(1520) is (24.92 ± 0.45 ± 0.48)%, while that of the resonance of mass of 1810 (or 1 820) MeV c−2 relative to Λ(1520) is (16.13 ± 0.38 ± 0.45)%. The branching ratio of the f2′(1525) resonance relative to φ0(1020) is (10.75 ± 0.25 ± 0.45)%. Data analysis was performed over 6 × 107 trigger events registered by the SELEX setup on the FNAL Tevatron.

Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing: Neutral SELEX

BackgroundSELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection.Methodology/Principal FindingsTo evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection.Conclusions/SignificanceOur data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.

Correlations of Λ hyperons with a small relative momentum in the SELEX experiment

The information on the shape of the potential between two baryons at small distances can be obtained by studying the baryon correlations at a small relative momentum in the creation reactions of high-energy hadron collisions. We have physically analyzed the SELEX experimental data and identified more than 20 × 103 events with creation of two Λ hyperons, which substantially exceeds the world statistical data. The obtained results are preliminary analyzed.

Software framework development for a physical analysis of SELEX experimental data

A software framework for a physical analysis of the results of experiments at a hadron spectrometer has been developed. This software is used to analyze the data of the SELEX experiment.

Cross-species de novo identification of cis-regulatory modules with GibbsModule: Application to gene regulation in embryonic stem cells

We introduce the GibbsModule algorithm for de novo detection of cis-regulatory motifs and modules in eukaryote genomes. GibbsModule models the coexpressed genes within one species as sharing a core cis-regulatory motif and each homologous gene group as sharing a homologous cis-regulatory module (CRM), characterized by a similar composition of motifs. Without using a predetermined alignment result, GibbsModule iteratively updates the core motif shared by coexpressed genes and traces the homologous CRMs that contain the core motif. GibbsModule achieved substantial improvements in both precision and recall as compared with peer algorithms on a number of synthetic and real data sets. Applying GibbsModule to analyze the binding regions of the Krüppel-like factor (KLF) transcription factor in embryonic stem cells (ESCs), we discovered a motif that differs from a previously published KLF motif identified by a SELEX experiment, but the new motif is consistent with mutagenesis analysis. The SOX2 motif was found to be a collaborating motif to the KLF motif in ESCs. We used quantitative chromatin immunoprecipitation (ChIP) analysis to test whether GibbsModule could distinguish functional and nonfunctional binding sites. All seven tested binding sites in GibbsModule-predicted CRMs had higher ChIP signals as compared with the other seven tested binding sites located outside of predicted CRMs. GibbsModule is available at (http://biocomp.bioen.uiuc.edu/GibbsModule).

Heavy-Heavy-Light Quark Potential in Two Approaches

There are a large number of studies about the interquark potential in hadrons. The interquark potential is mainly generated by gluon dynamics reflecting the SU(3) gauge symmetry. In particular, as many lattice QCD studies, the static and quenched approximation produces the only gluonic potential. However, realistic hadrons include not only gluons but also quarks, and quarks affect the interquark potential. We can consider two types of the quark effects, dynamical quark effects and valence quark effects. An example of the dynamical quark effects is the string breaking, which is obtained by unquenched calculation. In this study, we focus on the motional effects of finite-mass valence quarks, that is, valence quarks are not static but have a finite mass. To investigate such quark effects, we define the heavy-heavy-light (QQq) quark potential VQQq(R). It is defined as the energy of QQq systems in terms of the interheavy-quark distance R. The QQq system is constructed from two heavy quarks which are infinitely heavy and spatially fixed, and one light quark which is finite mass and moving around. Since this QQq potential effectively includes the lightquark motional effect, we can investigate the finite-mass valence quark effects to the interquark force. This QQq system is also related to the doubly charmed baryon, which has been recently discovered at the SELEX experiment.1),2) To calculate the QQq potential, we adopt the two approaches, SU(3) lattice QCD3) and a non-relativistic potential model.4) Lattice QCD is the first principle calculation based on QCD. The potential model, or the quark model, is one of the most successful analytical approaches for the low-energy hadron physics.

Complex Target SELEX

Aptamers are non-naturally occurring structured oligonucleotides that may bind to small molecules, peptides, and proteins. Typically, aptamers are generated by an in vitro selection process referred to as SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents. When the target protein requires the presence of the cell membrane (e.g., G-protein-coupled receptors, ion channels) or a co-receptor to fold properly, it is difficult or impossible to program the SELEX experiment with purified, soluble protein target. Recent advances in which the useful range of SELEX has been extended from comparatively simple purified forms of soluble proteins to complex mixtures of proteins in membrane preparations or in situ on the surfaces of living cells offer the potential to discover aptamers against previously intractable targets. Additionally, in cases in which a cell-type specific diagnostic is sought, the most desirable target on the cell surface may not be known. Successful application of aptamer selection techniques to complex protein mixtures can be performed even in the absence of detailed target knowledge and characterization. This Account presents a review of recent work in which membrane preparations or whole cells have been utilized to generate aptamers to cell surface targets. SELEX experiments utilizing a range of target "scaffolds" are described, including cell fragments, parasites and bacteria, viruses, and a variety of human cell types including adult mesenchymal stem cells and tumor lines. Complex target SELEX can enable isolation of potent and selective aptamers directed against a variety of cell-surface proteins, including receptors and markers of cellular differentiation, as well as determinants of disease in pathogenic organisms, and as such should have wide therapeutic and diagnostic utility.