To study RNA-peptide interactions, we performed an in vitro selection of RNA on a simple alpha-helical peptide-immobilized quartz-crystal microbalance (QCM) and evaluated the association constants (10(7) M-1) of the selected RNA to the model peptide on the same QCM plate.
To study RNA-peptide interactions, we performed an in vitro selection of RNA on a 27 MHz quartz-crystal microbalance (QCM) on which a simple R5 helix peptide was immobilized as a model of N peptide from bacteriophade X. The consensus sequences including a GNRA tetraloop were obtained from a random RNA pool after the 7 th cycle selection.
A simple α-helical N-model-peptide was designed to investigate the role of the arginine-rich motif of bacteriophage λ N-peptide in selective binding with boxB RNA. The five-arginine arrangement of native N-peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random-nucleotide region) was carried out with N-model-peptide immobilized on a 27 MHz quartz-crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (Ka =10(7) -10(8) M(-1) ). Many selected RNAs contained GNR(N)A-type loops (similar to the boxB RNA motif recognized by the native N-peptide). Fragments and minimal RNAs containing the GNRA-type loop also bound to N-model-peptide (Ka =10(6) -10(7) M(-1) ). The RNA recognition specificity of the peptide was studied by changing the "closing" U-A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N-model-peptide). It was concluded that the five-arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N-peptide adds the recognition ability for a loop-RNA sequence.
Quantitation of the level of specific mRNA involves the isolation of total RNA or poly(A)+ RNA as a starting materiaL Thus, this result is the sum of the transcription and degradation of mRNA. Here we report a rapid, sensitive, and high-throughput methodology for gene expression analysis from nuclear poly(A)+ RNA via the reduction of the cytosolic components. The cells were first trapped on the glass fiber membranes of 96-well filter plates and subsequently exposed to non-ionic detergent to achieve cell membrane permeation. The cytosolic components, which contain preexisting mRNA, were removed by washing with the appropriate buffer, while nuclei remained in the filter plates. Lysis buffer was then used to release nuclear mRNA, which was collected on oligo(dT)-immobilized PCR plates for the capture of poly(A)+ RNA, on which RT-PCR was performed. The reduction of the cytosolic components and the preservation of the nuclear components were confirmed by electron microscopy, agarose gel electrophoresis, PCR of mtDNA, and RT-PCR of pre-splicing immature beta-actin poly(A)+ RNA. Using this method, we clearly identified UVC-induced p21 gene expression that is not detectable with conventional whole cell methods.
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