Sialyllactose-binding modified DNA aptamer bearing additional functionality by SELEX

We produced a novel cationic-charged modified DNA aptamer for sialyllactose that is a ubiquitous component of the cell surface responsible for the infection of several viruses by using the magnetic-particle-based SELEX method. After 13 rounds of selection we selected 22 clones as sialyllactose-binding DNA aptamers composed of several modified thymidines. The DNA aptamers could form a three-way junction structure that likely forms a binding site for siallyllactose. The three-way junction structure contains several modified thymidines bearing a positively-charged amino group at the C5 position, which could enhance the binding ability for silalyllactose which has a negatively-charged carboxyl group. The dissociation constant of the aptamer that showed the strongest sialyllactose-binding ability among the clones of the aptamers was 4.9 microM.     

Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides

Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4.0–6.6 kcal/mol. Slight destabilization, by ∼0.5–1.5 kcal/mol, is observed for 5′- or 3′-terminal UNA residues. Furthermore, thermodynamic effects caused by UNA residues are extremely additive with ΔG°₃₇ conformity up to 98%. Direct mismatches involving UNA residues decrease the thermodynamic stability less than unmodified mismatches in RNA duplexes. Additionally, the presence of UNA residues adjacent to unpaired RNA residues reduces mismatch discrimination. Thermodynamic analysis of UNA 5′- and 3′-dangling ends revealed that stacking interactions of UNA residues are always less favourable than that of RNA residues. Finally, circular dichroism spectra imply no changes in overall A-form structure of UNA–RNA/RNA duplexes relative to the unmodified RNA duplexes.     

Study of a yeast mutant with modified mitochondrial translocation of adenine nucleotides

The effect of ethidium bromide on the growth of a yeast mutant with an impaired mitochrondrial translocation system of adenine nucleotides (op-1 mutant) was investigated. It was found that the op-1 mutant stops growing both under growing and non-growing conditions after treatment with ethidium bromide and that the growth cannot be restored by adding low-molecular compounds to the growth medium. It was the aim of the experiments to clarify whether the cessation of growth of the op-1 mutant after induction of the rho- mutation can be simulated by inhibitors phenotypically changing the mitochondrial function. It appears likely that the op-1 mutant stops growing only after the rho- mutation has been induced, because the phenotypic simulation of the rho- mutation does not lead the cessation of growth of the op-1 mutant.     

Study of a yeast mutant with modified mitochondrial translocation of adenine nucleotides

A yeast mutant with an impaired system of translocation of adenine nucleotides across the mitochondrial membrane, which stops dividing after superposition of the λ - mutation, was investigated. The results of this work indicate that combination of the op₁ mutation with the λ_- mutation in a single cell results in interruption of synthesis of polysaccharides and DNA leading to cessation of division of theop ₁λ mutant. The mechanism of this effect remains unclear.     

Probing the SELEX process with next-generation sequencing

Background

SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process.

Methodology

We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel.

Conclusions

High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.     

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

Background

SELEX 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 Findings

To 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/Significance

Our 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.     

SELEX技术与适体

SELEX技术是从随机寡核苷酸评议库中筛选与靶分子特异结合序列的组合化学技术。随机寡核苷酸的多种空间结构是筛选基础。该技术对靶分子无特殊要求,筛选出的寡核苷酸被称为适体,适体最突出的特征是与靶分子的特异性高亲和力。     

Ribose modified nucleosides and nucleotides as ligands for purine receptors

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.     

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19(+) Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.     

Libraries for genomic SELEX.

An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX.     

Rapid print-readout technique for sequencing of RNA's containing modified nucleotides.

A rapid, simple, and highly sensitive method for sequence analysis of RNA was developed, which consists of the following steps: (i) controlled hydrolysis of the RNA by brief heating in water; (ii) (32P)-labeling of 5'-hydroxyl groups of the fragments produced in (i); (iii) resolution of labeled fragments by size on polyacrylamide gels giving the familiar "ladder"; (iv) contact transfer ("print") of the ladder from the gel to a PEI-cellulose thin layer; (v) in situ treatment of the ladder with RNase T2 resulting in the release of 5'-(32P)-labeled nucleoside-3',5' diphosphates; (vi) contact transfer and thin-layer separation of (32P)-labeled nucleotides on PEI-cellulose in ammonium sulfate and ammonium formate solvents; (vii) autoradiography. The chromatographic behavior of the 4 major and 18 modified nucleotides was determined. The positions of major and modified nucleotides in the sequence can be read directly from the separation patterns displayed on X-ray film. As this is the only sequencing method presently available that allows one to display and identify directly the positions in the RNA chain of major and modified nucleotides, no additional procedures are required to analyze the latter.     

Nucleotide bias observed with a short SELEX RNA aptamer library

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20) with an abbreviated variable region. The Sel2N20 yields RNAs 51?nt in length, which unlike longer RNAs, are more amenable to large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine, resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in control, "nontargeted" selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or more of the amplification and propagation steps of the SELEX process.     

Lactoferrin interacts with nucleotides

Lactoferrin (LF) is an iron-binding glycoprotein found predominantly in milk and in granulocytes. LF is extremely polyfunctional protein some biological functions of which are determined by its capacity to bind iron, but many other functions are iron-independent. In this article we show for the first time that LF interacts with a number of various mononucleotides.     

SELEX and the evolution of genomes

The interrupted genome structures of complex multicellular organisms have most likely changed the evolution of the regulation of metabolism and development. Wasted intron sequences make regulation of gene expression in (for example) mammals appear to be unnecessarily complicated. The recent discoveries that globular RNA molecules are very much like the antigen-combining sites of antibodies suggest that intronic RNA may be used to help solve the problems raised by this complexity.     

SELEX技术应用研究进展

SELEX技术是一项在体外筛选能与各种配体特异结合的寡聚核苷酸片段的新组合化学技术。其优点是筛选出的核酸适体易合成、易存储、易修饰等。该技术不仅在疾病(如艾滋病)的治疗及药物的开发等方面起着重要的作用,也为核酸和蛋白质结构及功能的研究提供了一种有效的方法。     

A Computational Study of Alternate SELEX

Systematic evolution of ligands by exponential enrichment (SELEX) is a procedure for identifying nucleic acid (NA) molecules with affinities for specific target species, such as proteins, peptides, or small organic molecules. Here, we extend the work in Seo et al. (Bull Math Biol 72:1623–1665, 2010) (multiple-target SELEX or positive SELEX) and examine an alternate SELEX process with multiple targets by incorporating negative selection into a positive SELEX protocol. The alternate SELEX process is done iteratively by alternating several positive selection rounds with several negative selection rounds. At the end of each positive selection round, NAs are eluted from the bound product and amplified by polymerase chain reaction (PCR) to increase the size of the pool of NA species that bind preferentially to the given positive target vector. The enriched population of NAs is then exposed to the negative targets (undesired targets). The free NA species (instead of the bound NA species being eluted) are retained and amplified by PCR (negative selection). The goal is to minimize an enrichment of nonspecifically binding NAs against multiple targets. While positive selection alone results in a pool of NAs that bind tightly to a given target vector, negative selection results in the subset of the NAs that bind best to the nontarget vectors that are also present. By alternating the two processes, we eventually obtain a refined population of nucleic acids that bind to the desired target(s) with high “selectivity” and “specificity.” In the present paper, we give formulations of the negative and alternate selection processes and define their efficiencies in a meaningful way. We study the asymptotic behavior of alternate SELEX system as a discrete-time dynamical system. To do this, we use the chemical potential to examine how alternate SELEX leads to the selection of NAs with more specific interactions when the ratio of the number of positive selection rounds to the number of negative selection rounds is fixed. Alternate SELEX is said to be globally asymptotically stable if, given the initial target vector and a fixed ratio, the distribution of the limiting NA fractions does not depend on the relative concentrations of the NAs in the initial pool (provided that all of the NA species are initially present in the initial pool). We state conditions on the matrix of NA—target affinities that determine when the alternate SELEX process is globally asymptotically stable in this sense and illustrate these results computationally.