In this paper, we refer to the complementary strands for hybridization to bound substrate probe DNA as target DNA and RNA. Selection of the 26-mer RNA sequence used here was based on in silico analysis of the TMV genome sequence simulating ribonuclease T1 (RNase T1) digestion (cleavage after G residues) followed by sorting of the fragments by size. RNase T1 digestion was selected because it can yield a population of RNAs with more controlled sizes defined by the distribution of G residues within the degraded RNA. The 26-mer TMV sequence used was chosen both for its length and GC content. It is also sufficiently long that it is unlikely that its sequence would be found in the transcriptosome of uninfected plants and presumably is long enough for stable hybridization at room temperature or above.
The DNA probe, a thiolated 26 nucleotide precursor (SH-26-mer) (provided as the disulfide), and the target sequence RNA and DNA 26-mers used in this study were synthesized by Integrated DNA Technologies (Coralville, Iowa). Their sequences are as follows.