ABSTRACT
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.
Subject(s)
Bacteriophages , Capsid Proteins , Capsid/metabolism , Genome, Bacterial , RNA, Bacterial/metabolism , RNA-Binding Proteins/metabolism , Artifacts , Base Sequence , Binding Sites , Computational Biology , Consensus Sequence , Genes, Bacterial/genetics , Genomic Library , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Polymerase Chain Reaction , Protein Binding , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Sensitivity and Specificity , Substrate Specificity , Transcription, GeneticABSTRACT
T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4 messages, rendering them nonfunctional. Not all -GGAG- sites are processed equally by RegB; those found at the Shine-Dalgarno sequences and in intercistronic regions are processed with higher efficiency than the -GGAG- sites located in coding regions. The low activity of RegB observed in vitro is enhanced by 1-2 orders of magnitude by the Escherichia coli ribosomal protein S1. We have used SELEX (systematic evolution of ligands by exponential enrichment) on a combinatorial RNA library to obtain molecules that are specifically cleaved by T4 RegB endonuclease in the presence of S1. The sequences obtained contain the required -GGAG- tetranucleotide and were unusually enriched in adenosine and cytosine nucleotides. No consensus structure or sequence motif other than -GGAG- was conserved among the selected molecules. The majority of the RNAs are entirely dependent on S1 for RegB-catalyzed cleavage; however, a few RNAs are found to be S1 independent but are cleaved by RegB with much lower rates.
Subject(s)
Bacteriophage T4/metabolism , Endoribonucleases/metabolism , RNA/metabolism , Base Sequence , DNA Primers , Hydrolysis , Molecular Sequence Data , Nucleic Acid Conformation , RNA/chemistry , RNA Processing, Post-Transcriptional , Substrate SpecificityABSTRACT
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.
Subject(s)
Genomic Library , Nucleic Acids/metabolism , Protein Binding/genetics , Binding Sites/genetics , DNA, Bacterial/metabolism , DNA, Fungal/metabolism , Escherichia coli/genetics , Gene Dosage , Humans , Placenta/chemistry , Polymerase Chain Reaction , Saccharomyces cerevisiae/geneticsABSTRACT
The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?