RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis

Abstract

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.

Figure 1

Scheme depicting assay design for comparative sequence analysis by base-specific RNA cleavage reaction. Promotor sequences of T7 and SP6 RNA polymerase are tagged at the PCR primers adjacent to the target region. For post-PCR amplification, the sample is split and forward and reverse strands of the PCR product are transcribed into RNA by T7 and SP6 RNA polymerases, respectively. G-specific RNA cleavage reaction is performed by RNase T1 and obtained fragments are analyzed by MALDI-TOF MS.

Figure 2

MALDI-TOF fragmentation spectrum of ApoB-100 target region. RNA in-vitro transcription was performed with T7 RNA polymerase utilizing unmodified NTPs. Each mass signal is labeled with its reference number and was assigned to the corresponding RNA fragments calculated in silico (see Table 1). The masses of the individual fragments indicate a complete RNase cleavage reaction because all RNA fragments except for the 3′ terminal ones possess only a 5′-hydroxy and 3′-phosphate group.

Figure 3

Reproducibility of comparative sequence analysis via base-specific RNase T1 cleavage reaction. The G-specific cleavage patterns of six independent reactions of the ApoB-100 target region are shown. All samples were processed with automated liquid handling devices on different days with several individuals of the same sequence and genotype.

Figure 4

During the RNA in vitro transcription either U or C was replaced by its 5-methyl ribonucleotide triphosphate analog to circumvent the small mass difference between U and C that inhibits the differentiation between individual RNA fragments. (A) A subview into the base-specific cleavage spectrum of ApoB-100 target region where the transcription was performed with normal CTP and UTP. Three relevant signals at m/z = 2245.8, 2526.3 and 2905.0 occur. A G-specific cleavage spectrum of the same ApoB target is shown in the middle spectrum (B) where UTP is replaced by 5-Me UTP during the transcription reaction. The mass difference to the relevant original fragment is labeled and indicates the number of U bases within this fragment. (C) The result on the fragment masses if CTP is replaced by its Me-CTP analog. For both base modifications, each previous unresolved signal was separated into two individual peaks.

Figure 5

G-specific cleavage reactions applied to the I405V polymorphism of the CETP gene, analyzed in three different individuals. RNA transcription with T7 RNA polymerase was performed with unmodified NTPs. Unique signals corresponding to the individual genotype are marked with a vertical dotted line.

Figure 6

G-specific cleavage reactions applied to a polymorphism of the UCP2 gene, analyzed in three different individuals. RNA transcription was performed with SP6 RNA polymerase using unmodified NTP. Shown is a sub-view into the full spectrum. The abundance of a unique signal at m/z = 1304.8 corresponds to the individual genotype.

Figure 7

Overlay of three fragmentation mass spectra of different Bordetella strains (16S rDNA). RNA in vitro transcription was performed with SP6 RNA polymerase wherein CTP was replaced by Me-CTP. RNase T1 fragmentation reaction of 16S rDNA of yet-uncultured rDNA clone SHA 110 (upper spectrum), bacterium B-52 (middle spectrum) and Bordetella trematum (lower spectrum). Unique signals, marked by an arrow, were used for strain identification.