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.     

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies ¹. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules ^2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process ³, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection ⁵. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX ⁶, or they have been trimmed to 6-9 nt ^{7, 8}. Wen and Gray ⁹ designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification ¹⁰.We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems ^{11, 12}. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10^-7 - 10^-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.Download video file.^{(128M, mp4)}     

Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX

Genomic SELEX is a method for studying the network of nucleic acid–protein interactions within any organism. Here we report the discovery of several interesting and potentially biologically important interactions using genomic SELEX. We have found that bacteriophage MS2 coat protein binds several Escherichia coli mRNA fragments more tightly than it binds the natural, well-studied, phage mRNA site. MS2 coat protein binds mRNA fragments from rffG (involved in formation of lipopolysaccharide in the bacterial outer membrane), ebgR (lactose utilization repressor), as well as from several other genes. Genomic SELEX may yield experimentally induced artifacts, such as molecules in which the fixed sequences participate in binding. We describe several methods (annealing of oligonucleotides complementary to fixed sequences or switching fixed sequences) to eliminate some, or almost all, of these artifacts. Such methods may be useful tools for both randomized sequence SELEX and genomic SELEX.     

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.     

In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries.

The Epstein-Barr virus (EBV)-expressed RNA 1 (EBER1) associates tightly with the ribosomal protein L22. We determined the general requirements for an RNA to bind L22 in a SELEX experiment, selecting RNA ligands for L22 from a randomized pool of RNA sequences by using an L22-glutathione S-transferase fusion protein. The selected sequences all contained a stem-loop motif similar to that of the region of EBER1 previously shown to interact with L22. The nucleotides were highly conserved at three positions within the stem-loop and identical to the corresponding nucleotides in EBER1. Two independent binding sites for L22 could be identified in EBER1, and mobility shift assays indicated that two L22 molecules can interact with EBER1 simultaneously. To search for a cellular L22 ligand, we constructed a SELEX library from cDNA fragments derived from RNA that was coimmunoprecipitated with L22 from an EBV-negative whole-cell lysate. After four rounds of selection and amplification, most of the clones that were obtained overlapped a sequence corresponding to the stem-loop between nucleotides 302 and 317 in human 28S ribosomal RNA. This stem-loop fulfills the criteria for optimal binding to L22 that were defined by SELEX, suggesting that human 28S ribosomal RNA is likely to be a cellular L22 ligand. Additional L22 binding sites were found in 28S ribosomal RNA, as well as within 18S ribosomal RNA and in RNA segments not present in sequence databases. The methodology described for the conversion of a preselected cellular RNA pool into a SELEX library might be generally applicable to other proteins for the identification of cellular RNA ligands.     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.     

变形链球菌反应调控蛋白ComE结合序列的初步筛选

目的：从变形链球菌基因组中筛选反应调控蛋白ComE结合的核酸序列。方法：⑴提取变形链球菌UA159 基因组,进行超声剪切处理。以基因组片段为模板,用随机引物进行延伸,切胶纯化和PCR 扩增后获得基因组文库。⑵应用基因组 SELEX 技术,以ComE 蛋白为靶物质,与变形链球菌基因组文库共同孵育,循环筛选8轮。筛选产物TA克隆后送去测序,测序结果进行生物信息学分析。⑶将分析得到的2个序列分别克隆入pFW5-luc载体中,然后分别重组到变形链球菌UA159野生株和comE突变株的基因组中,采用RT-PCR的方法检测luc片段的表达。结果：⑴获得了变形链球菌UA159 的基因组文库,片段范围约为100～300bp。⑵随机挑取54个克隆送去测序,经生物信息学分析和RT-PCR初步验证,最终获得1个ComE 蛋白可能的结合序列。结论：采用基因组 SELEX 技术,筛选反应调控蛋白ComE可能的结合序列,并进行了初步验证,为后续进行变形链球菌反应调控蛋白ComE调控机制的研究奠定了基础。     

Mimotopes and proteome analyses using human genomic and cDNA epitope phage display

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 x 106 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as 'mimotopes' (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences.ABBREVIATIONS USED: IL, interleukin; ELISA, enzyme linked immunoabsorbant assay; PBS, phospho-buffered saline; cfu, colony forming units.     

Restriction cutting independent method for cloning genomic DNA segments outside the boundaries of known sequences

We present a simple method for cloning genomic DNA segments outside the boundaries of known sequences, which is not dependent on restriction cutting or mapping. In the first step of the method, a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are then amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally, the different fragments are separated by cloning and characterized by sequencing. The method was used to clone both the upstream (5') and the downstream (3') genomic regions of an intron-interrupted tRNA(Leu)(UAA) gene from three cyanobacteria belonging to the genus Microcystis.     

A new technique for selective identification and mapping of enhancers within long genomic sequences

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.     

The genomic organization of the rat AT1 angiotensin receptor.

The AT1 receptor subtype modulates all of the hemodynamic effects of the vasoactive peptide, angiotensin II. In this report, we investigate the genomic organization of this important receptor. A rat genomic library was screened with fragments from the 5' region of a previously cloned cDNA, pCa18b, encoding the rat AT1 receptor. Two lambda clones were isolated and the hybridizing restriction fragments were sequenced. Comparison of the genomic and cDNA sequences reveals that the rat AT1 receptor has three exons. Two of the exons encode 5' untranslated sequence while the third exon encompasses the entire coding region, a small portion of the 5' untranslated region and the entire 3' untranslated sequence. Further analysis of the genomic sequence 5' to the start site of pCa18b demonstrates typical sequence motifs found in many eukaryotic promoters including a TATA box, a cap site and a potential Sp1 binding site. Southern analysis of genomic DNA indicates that the AT1 receptor subtype represented by pCa18b is encoded by one gene within the rat genome.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).     

Identification of CHO Endogenous Gene Regulatory Elements

The objective of this approach was to identify new CHO endogenous gene regulatory elements that are capable of regulating foreign gene expression in recombinant CHO host cells. The standard technology for the production of many biopharmaceutical products is frequently based on expression vectors that utilize strong mammalian viral promoters like SV40 or CMV which allow for very high expression rates but this may lead to constitutive over-expression resulting in a permanent stress for the cell. In addition, some heterologous promoters are cell-cycle dependent and can be subject to gene silencing generating heterogeneity within the cell population. Here, we describe the construction of a genomic CHO library and the subsequent identification and isolation of selected target sequences that are believed to be responsible for high level expression of the associated genes. The method that was used to isolate these regions of interest relies on gene specific amplification with primer pairs binding on different genes and the vector sequence. Flanking regions of these fragments were identified through Inverse PCR from fragmented and self-ligated genomic DNA. Expression levels of both the initially derived and the mapped fragments were determined through a luciferase reporter assay.     

SELEX selection of high-affinity oligonucleotides for bacteriophage Ff gene 5 protein

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.     

Cloning genomic sequences using long-range PCR

Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described.     

用于高通量测序的基因组靶序列捕获方法的建立

文章旨在建立一种基因组目标靶序列捕捉文库的方法,并结合第二代测序技术,以实现候选基因区段的深度测序。利用Agilent公司的eArray在线平台,对1250个基因的11824个外显子共2414977bp的基因组序列进行120个碱基长度的捕捉探针(钓饵)设计,并制备成SureSelect液相靶序列捕获试剂。选用2例人基因组DNA,超声打断后末端补平并磷酸化,连接SOLiD接头,回收150bp～200bp的DNA片段,与靶序列探针杂交捕获目标序列,油包水微乳滴PCR扩增后,磁珠分离富集,上SOLiD测序系统通过工作流程分析(WFA)进行文库质量的评价,或正式测序反应。结果显示对所包含的11147个基因外显子片段设计出并合成了46509个捕捉探针,制备成SureSelect试剂盒。探针可有效地捕捉并富集基因组DNA的目标靶片段,定量PCR显示富集效率可达29倍。WFA分析表明文库可以在SOLiD仪器进行正式测序。测序结果显示靶序列区域的测序数占有效总测序数的比例达到70%,覆盖率均在200×以上。结果表明本研究所建立的SureSelect基因组靶序列捕捉、富集建立测序文库的技术路线可行,可直接用于SOLiD测序仪的测序。     

One-base excess adaptor ligation method for walking uncloned genomic DNA

This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5′-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5′-overhang base overlaps the corresponding 3′-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3′-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5′-terminal base was not consistent with the corresponding 3′-terminal base. Using this technique, the 3′- and 5′-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.