Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I.

RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5'-end (RNA I-5) resulted in an approximately twofold increase in the steady-state levels of RNA I-5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These results indicate that RNA I-5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5'-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5'-monophosphorylated end.

Development of DNA aptamers specific for small therapeutic peptides using a modified SELEX method.

Aptamers are short single-stranded DNA or RNA oligonucleotides capable of binding with high affinity and specificity to target molecules. Because of their durability and ease of synthesis, aptamers are used in a wide range of biomedical fields, including the diagnosis of diseases and targeted delivery of therapeutic agents. The aptamers were selected using a process called systematic evolution of ligands by exponential enrichment (SELEX), which has been improved for various research purposes since its development in 1990. In this protocol, we describe a modified SELEX method that rapidly produces high aptamer screening yields using two types of magnetic beads. Using this method, we isolated an aptamer that specifically binds to an antimicrobial peptide. We suggest that by conjugating a small therapeutic-specific aptamer to a gold nanoparticle-based delivery system, which enhances the stability and intracellular delivery of peptides, aptamers selected by our method can be used for the development of therapeutic agents utilizing small therapeutic peptides.