Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.     

Pitfalls of cell-systematic evolution of ligands by exponential enrichment (SELEX): existing dead cells during in vitro selection anticipate the enrichment of specific aptamers

Aptamers represent auspicious ligands for recognition of target molecules on the surface of a specific cell population, such as stem or cancer cells. These ligands are able to capture and enrich desired cells from a cell mixture, and can be used for identification of new biomarkers, development of cell-specific therapeutics, and stem cell therapy. In this study, we investigated the influence of dead cells on single-stranded DNA (ssDNA) binding and established a method to eliminate dead cells from a cell suspension. Flow cytometry analyses demonstrated that all dead cells were stained with fluorescein-labeled ssDNA molecules. The increasing of the proportion of dead cells led to an increased number of cells that were positive for ssDNA staining. Using dead cell removal microbeads, the proportion of dead cells was significantly reduced. The studies demonstrated that dead cells lead to unspecific uptake/binding of ssDNA molecules during cell-Systematic Evolution of Ligands by Exponential enrichment (SELEX) and can cause failure of the selection process. Thus, the elimination of dead cell population before incubation with ssDNA molecules will reduce the loss of target binding sequences and the contamination of the enriched aptamer pool with unspecific ssDNA molecules caused by unspecific binding to dead cells.     

化学修饰寡核苷酸在核酸配基扩增技术中的应用

核酸酸基扩增技术（SELEX)可从极大容量的随机寡核苷酸文库中筛选得到与靶分子高特异性和高亲和力结合的核酸配基。对寡核苷酸进行化学修饰,可以提高核酸配基的稳定性,增加其功能多样性及生物利用度。SELEX在基础研究、诊断和治疗试剂的研制及药物筛选等领域有广泛用途。     

DNA aptamers specific for Legionella pneumophila: systematic evolution of ligands by exponential enrichment in whole bacterial cells

Biotechnology Letters - Legionella pneumophila is the major causative agent of Legionnaires’ disease and Pontiac fever, which pose major public health problems. Rapid detection of L....     

Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment

ETR-3 (also know as BRUNOL3, NAPOR, and CUGBP2) is one of six members of the CELF (CUG-BP1- and ETR-3-like factor) family of splicing regulators. ETR-3 regulates splicing by direct binding to the pre-mRNA. We performed systematic evolution of ligands by exponential enrichment (SELEX) to identify the preferred binding sequence of ETR-3. After five rounds of SELEX, ETR-3 selected UG-rich sequences, in particular UG repeats and UGUU motifs. Either of these selected motifs was able to restore ETR-3 binding and responsiveness to a nonresponsive splicing reporter in vivo. Moreover, this effect was not specific to ETR-3 since minigenes containing either of the two motifs were responsive to two other CELF proteins (CUG-BP1 and CELF4), indicating that different members of the CELF family can mediate their effects via a common binding site. Using the SELEX-identified motifs to search the human genome, we identified several possible new ETR-3 targets. We created minigenes for two of these genes, the CFTR and MTMR1 genes, and confirmed that ETR-3 regulates their splicing patterns. For the CFTR minigene this regulation was demonstrated to be dependent on the presence of the putative binding site identified in our screen. These results validate this approach to search for new targets for RNA processing proteins.     

Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment

In this paper, single-stranded (ss)DNA aptamers with capability to distinguish differentiated PC12 cells from normal PC12 cells were selected by subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. Before each round of selection, randomized ssDNAs were incubated with regular PC12 cells to eliminate those that recognize the common cellular components of both differentiated and undifferentiated PC12 cells. After six rounds of cell-based selection, both of individual aptamers and aptamers of the sixth round pool were found binding to differentiated PC12 cells, but not to the parental PC12 cells. The aptamers of the starting pool showed no such binding. Sequence analysis illustrated that the amount of G content in central random region of these aptamers was much higher than that of the starting pool, which would be expected to be average. The aptamers obtained from this method were also able to identify differentiated PC12 cells from a mixture of both normal and differentiated cells. The results indicate that subtractive SELEX is a useful tool in finding ligands to specific biological markers that distinguish a subtype of cells from cells of homologous origin, such as carcinoma cells among normal epithelial tissues. Both these aptamers and their markers may play important roles in basic research and clinical diagnosis.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.     

SELEX for tubulin affords specific T-rich DNA aptamers. Systematic evolution of ligands by exponeential enrichment.

We succeeded in acquiring two DNA aptamers that selectively recognize tubulin by the SELEX method. A pool of single-stranded oligo-DNAs including a random region of 59 nucleotides was screened by SELEX for tubulin purified from calf-brain as a target. After 20 repetitions of selection round, the library converged on specific T-rich sequences. The binding activity of T-rich clones was analyzed by the SPR sensor to determine their dissociation constants to be in the order of 10 microM.