/pdf/functional-nucleic-acid-sensors-k8tlieshyr.pdf

Functional Nucleic Acid Sensors

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands.

Analytical applications of aptamers.

Aptamers are single-stranded nucleic acid molecules that possess properties comparable to those of protein monoclonal antibodies, and thus are clear alternatives to long established antibody-based diagnostic or biotechnological products for research, diagnostics, and therapy. 1-4 This class of functional nucleic acids can fold into complex three-dimensional shapes, 5-7 forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target, 8-10 from metal ions and small chemicals to large proteins and higher order protein complexes, whole cells, viruses, or parasites. Aptamers can be isolated in Vitro from vast combinatorial libraries that comprise trillions of different sequences, by a process called “ in Vitro selection”, or “SELEX”, an acronym for “systematic evolution of ligands by exponential enrichment”. An in Vitro selection experiment comprises a number of sequential steps, the first of which is the generation of a nucleic acid library of random sequences. This starting pool of mainly nonfunctional RNA or DNA sequences is generated using a standard DNA-oligonucleotide synthesizer. 11 The design of such libraries involves the synthesis of a short defined sequence, followed by a random region of variable length and another defined sequence at the 5 ′-end. This pool of synthetic single-stranded DNA (ssDNA) is amplified in the polymerase chain reaction (PCR), generating several copies of each DNA in its double-stranded form. By in Vitro transcription, a corresponding library of RNA molecules can be generated which can then be used for the in Vitro selection. If the transcription reaction contains nucleoside triphosphate derivatives that are chemically modified but still are substrates for RNA polymerases, libraries of modified RNAs can be generated in which sequences are equipped with a broad variety of additional chemical functionalities, normally not present in natural nucleic acids. 12-14 Aptamers selected from chemically modified libraries can, in some cases, be completely resistant toward degradation by nucleases. The additional functional groups can lead to ligands with novel * To whom correspondence should be addressed: telephone, +49-228735661; fax,+49-228-735388; e-mail, m.famulok@uni-bonn.de. † LIMES Institute. ‡ University of Konstanz. Volume 107, Number 9

Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy

In the past two decades, high-affinity nucleic acid aptamers have been developed for a wide variety of pure molecules and complex systems such as live cells. Conceptually, aptamers are developed by an evolutionary process, whereby, as selection progresses, sequences with a certain conformation capable of binding to the target of interest emerge and dominate the pool. This protocol, cell-SELEX (systematic evolution of ligands by exponential enrichment), is a method that can generate DNA aptamers that can bind specifically to a cell type of interest. Commonly, a cancer cell line is used as the target to generate aptamers that can differentiate that cell type from other cancers or normal cells. A single-stranded DNA (ssDNA) library pool is incubated with the target cells. Nonbinding sequences are washed off and bound sequences are recovered from the cells by heating cell-DNA complexes at 95 degrees C, followed by centrifugation. The recovered pool is incubated with the control cell line to filter out the sequences that bind to common molecules on both the target and the control, leading to the enrichment of specific binders to the target. Binding sequences are amplified by PCR using fluorescein isothiocyanate-labeled sense and biotin-labeled antisense primers. This is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection. The enrichment of the selected pools is monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library. The procedure, from design of oligonucleotides to enrichment of the selected pools, takes approximately 3 months.

Development of DNA aptamers using Cell-SELEX

One of the key components of proteomics initiatives is the production of high affinity ligands or probes that specifically recognize protein targets in assays that detect and capture proteins of interest. Particularly versatile probes with tremendous potential for use as affinity molecules are aptamers. Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on affinity for a target molecule. Aptamers offer advantages over traditional antibody-based affinity molecules in their ease of production, regeneration and stability, largely due to the chemical properties of nucleic acids versus amino acids. We describe an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. We demonstrate the ability of aptamers that recognize thyroid transcription factor 1 (TTF1) to bind their target protein with high affinity and specificity, and detail their uses in a number of assays. The TTF1 aptamers were characterized using surface plasmon resonance, and shown to be useful for enzyme-linked assays, western blots and affinity purification.

/pdf/an-improved-method-for-the-in-vitro-evolution-of-aptamers-32v2eg39xx.pdf

An improved method for the in vitro evolution of aptamers and applications in protein detection and purification

Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein.

Summary

The fluffy (fl) gene of Neurospora crassa is required for asexual sporulation and encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA-binding motif. Identification of genes regulated by fl will provide insight into how fungi regulate growth during morphogenesis. As a step towards identifying the target genes on which FL may act, we sought to define target sequences to which the FL protein binds. The DNA binding domain of FL was expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified using glutathione-sepharose affinity chromatography. The DNA binding sites were selected and amplified by means of a polymerase chain reaction (PCR)-mediated random-site selection method involving affinity bead-binding and gel mobility shift analysis. Sequencing and comparison of the selected clones suggested that FL binds to the motif 5′-CGG(N)9CCG-3′. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5′-CGGAAGTTTC CTCCG-3′, which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag also confirmed that this sequence resides in a region required for FL regulation. In addition, yeast one hybrid experiments demonstrated that the C-terminal portion of FL functions in transcriptional activation. Transcriptional profiling was used to identify additional potential targets for regulation by fl.

Fluffy, the major regulator of conidiation in Neurospora crassa, directly activates a developmentally regulated hydrophobin gene.

Author(s): Murphy, Michael B.; Doyle, Sharon A. | Abstract: Large collections of purified proteins have become essential to systems biology programs for generating protein ligands and for validating protein interactions. These large protein collections will likewise benefit structural studies, and quests for protein-based therapeutics. Whether many protein targets need to be produced, such as a microbial proteome, or a few poorly expressing protein targets need to be expressed as soluble fragments (divide and conquer approach), efficient high-throughput processing can be a bottleneck (1). This chapter describes a method for efficient high-throughput purification of hexahistidine-tagged proteins that are expressed in Eschericia coli (E. coli) using immobilized metal affinity chromatography (IMAC) (2) in a 96-well format. This approach is particularly suitable for proteomic applications that require modest amounts of highly purified proteins to be generated very efficiently. This approach is also very useful for identifying protein targets that are most amenable to scaled-up production for use in structural studies. The typical yield of proteins purified using this system is 50 150 micrograms, which is generally greater than that of many in vitro expression systems and much less costly. The method as described has been optimized for purifying approximately 150 micrograms of hexahistidine-tagged protein, but the method is flexible so that the amount of affinity matrix and culture volumes can be adjusted for optimal binding capacity and consequently highest purity. Although the method detailed here uses IMAC to purify hexahistidine-tagged proteins, this basic platform can be used with many other tags and affinity resins.

/pdf/high-throughput-purification-of-hexahistidine-tagged-1zhd0hsflq.pdf

High-throughput purification of hexahistidine-tagged proteins expressed in E. coli.

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Papers

An improved method for the in vitro evolution of aptamers and applications in protein detection and purification

Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein.

Fluffy, the major regulator of conidiation in Neurospora crassa, directly activates a developmentally regulated hydrophobin gene.

High-throughput purification of hexahistidine-tagged proteins expressed in E. coli.