RNA Polymerase Activity Assay on Biochips: Correlation between Template DNA Density and RNA Synthesis

Abstract

EDC: T-SH template DNA (B)T-SH template DNA 5'SH-AAC TGG CCG CTGAAG GGC TTT TGA ACT CTG CTT AAA TCC TAT AGT GAG TCG TAT TAG TCC-3' T7 primer 5'-GGACTAATACGACTCACTATA-3' Primer T7 RNA polymerase Figure 1. (A) Structure of SAMs on gold and chemical modifications to prepare template DNA-presenting biochips. The carboxylic acidpresenting monolayer was treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-aminoethyl maleimide, followed by thiolated template DNA. (B) Strategy for T7 RNAP activity assay on biochips and the sequences of oligonucleotides (T7 promoter sequences underlined) used in this study.In vitro synthesis of RNAs by bacteriophage T7 RNA polymerase (T7 RNAP) has been applied extensively to the investigation of RNA serving as a biologically active molecule. T7 RNAP is a single subunit enzyme with a molecular weight of 98 kDa that is capable of catalyzing transcription without any accessory proteins.1-3 Other RNA polymerases derived from bacteriophage (SP6 and T3) were also useful for in vitro synthesis of RNA. Each RNA polymerase has different DNA promoter sequences for initiation of transcription. The behavior of T7 RNAP is critically dependent on its interaction with the promoter element in template DNA. The T7 bacteriophage genome contains a variety of promoters that are recognized by T7 RNAP, all of which are related to a 23-base pair consensus sequence (see Figure 1). 1 The promoter can be divided into a recognition domain, encompassing positions 17 through 5, and an initiation domain, encompassing positions 4 through +6 with a transcription start site initiates at the +1 position. 4,5a These authors equally contributed to this study.Cell-free transcription and translation system on solid surface has been developed and utilized in many genomic studies.6-8 As a new research tool, cell-free gene expression has been applied to DNA-based biochips, which showed possibilities of developing synthetic biological systems and biochemical materials at nanoscale. [8][9][10][11] The biosynthetic reaction on surface of microchips was first realized by Buxboim et al. and they developed and utilized the microchips assembled by photolithographic approach on silicon dioxide (SiO2) for controlling the cell-free expression. 7,8,12 This approach allows us to control the immobilized DNA and regulate the biosynthetic reactions in a more controlled manner. 7 Recently, it has been reported that there exists attenuation of protein synthesis in a translation coupled with a transcription system on DNA-grafted biochips, which is likely due to molecular crowding caused by the translational machinery. 7Here, we investigated T7 RNAP enzyme activity of RNA synthesis on self assembled monolayers (SAMs) on gold surface which presents template DNA at various surface densities ranging from 5% to 50%. Using the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), binding of T7 RNAP to T7 promoter sequence in the DNA template on chip was analyzed and the activity of...

Analysis of chemical/biochemical conversions on gold microparticles using MALDI-TOF MS

“…While such demand has been achieved on two dimensional surfaces by fluorescence, electrochemistry, XPS, etc, no efficient methods for use on three dimensional surfaces have been reported 7,8 . Recently, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), which directly provides information regarding surface compositions, has been actively used for surface analysis of self-assembled monolayers on gold [9][10][11][12][13] . In this report, we extended this strategy to three dimensional surfaces.…”