We have synthesized a series of 5′-phosphorylated and 5′-cytidylyl-(3′–5′)-cytidylyl-(3′–5′)-puromycin derivatives that have backbone-elongated substrates. All the synthesized puromycin derivatives showed good solubility in water and were applied to translation inhibitory assay in a reconstituted Escherichia coli translation system.The synthesis of puromycin derivatives with backbone-elongated substrates is reported.

The “light-up” RNA aptamer–Hoechst pair can be used as a fluorescent tag to monitor transcription processes.

Using “chemically misacylated AMP” which is photogeneratable, tRNA can be enzymatically acylated with N-methylamino acids in an efficiency comparable to that of the corresponding natural aminoacylation process with amino acids.

The balance of the loop and the stem in terms of base-pair (bp) length is critical for sensitivity and selectivity. If the stem is long, the hairpin structure will be more stabilized and will be opened only by a longer target capable of more extensive target-probe hybridization. This will give a high sensitivity—that is, high target-on/target-off signal ratio—but low sequence selectivity. If, on the other hand, the stem is short, the hairpin structure will be less stabilized and easily opened even by a shorter target. This will give high sequence selectivity but a low sensitivity. Actually, we chose the commonly encountered 6-base AGGAGA sequence as RBS and set, after trial and error, an 8-bp stem and 18- or 16-bp target-probe hybridization at the loop to optimize sensitivity and selectivity.Another merit of the present system is that the reporter protein can, in principle, be of any type. This is particularly important in terms of single nucleotide polymorphism (SNP) detection, since we can easily construct a set of allele-sensitive probes using a class of otherwise closely related reporter proteins that can be distinguished from each other.Luciferase was our first choice as sensing output because of its high sensitivity, the good linearity of the related chemiluminescence assay and the ready color tuning (see below). Although this system requires luciferin as an external assay reagent, the latter is readily taken up into the cell and can thus be used for in vivo sensing20. A prototypical 18C-targeting MB-mRNA probe, designated G-RNABEST (Fig. 3), was obtained by sequential PCRs on the luciferase gene (luc) encoded in plasmid vector pBESTluc and subsequent transcription (Fig. 4). The first PCR added the RBS (red), an 18-nt target-binding site (blue) with a target-complementary G base, and an intervening spacer. The second PCR added an anti-RBS domain (pink) and an additional hairpin structure preceded by the T7 promoter region. The primer sequences used are summarized in Figure 5. For sequence-selective production of multicolor reporter proteins, we took advantage of the MultiReporter Assay System. Three plasmid vectors, pSLG-test, pSLO-test and pSLR-test, encode green-luminescing wide-type Rhagophthalmus ohbai firefly luciferase (G-luc; emission maximum, 550 nm), orange-luminescing mutated (T226N) R. ohbai firefly luciferase (O-luc; 580 nm) and red-luminescing wild-type Phrixothrix hirtus firefly luciferase (R-luc; 630 nm), respectively21, 22, 23. They were PCR-amplified as above to incorporate an A-sensitive, T-sensitive or G-sensitive target-binding site, respectively, to give U-RNASLG, A-RNASLO and C-RNASLR (Fig. 3). We also prepared an 18C-targeting probe, G-DNAgal (Fig. 3), encoding β-galactosidase (β-gal). The latter cleaves o-nitrophenyl β-galactoside as a substrate to give colored o-nitrophenol for colorimetric or visual assay.To further enhance catalytic performance, we also use RNase H. The translation-activated mRNA probe reversibly formed from stoichiometric binding of the target to the probe (structure on the right-hand side of Fig. 1b) is, at best, equimolar to the target. This would lead to low signal intensity, especially when the target &1QJ;is present in a tiny amount. RNase H is an endonuclease that specifically cleaves RNA in the DNA/RNA heteroduplex24. It thus irreversibly digests the target-bound loop region of the probe to release the anti-RBS domain from the mRNA body, thus allowing catalytic use of target DNA (RNase H–coupled MB-mRNA; Fig. 1c) with improved selectivity and enhanced sensitivity25, although, given the nature of RNase H, it can be used only in vitro for DNA targets and not for in-cell gene sensing, where the targets should be mRNAs.The translation- or transcription/translation-coupled sensing of ODN targets can be carried out in a normal Escherichia coli S30 extract system such as RTS HY100 (Roche), but we use a reconstituted prokaryotic cell–free transcription/translation system (Pure System; Post Genome Institute)26 throughout this work. Colorimetric assay of β-galactosidase cannot be conducted in the E. coli extract, which itself contains the enzyme. The effect of RNase H can also be most clearly evaluated in the reconstituted medium, which is otherwise free from RNase H.For negative control, add 1 μl water in place of target 18C.For negative control, add 1 μl water in place of target ODN.For negative control, add 1 μl water in place of target 18C.For negative control, add 1 μl water in place of target ODN.For negative control, add 1 μl water in place of target 18C.Troubleshooting advice can be found in Table 1.In the case of short 16-nt targets, even 16C (I on/I off = 1.4; lane 9), in addition to 16T (I on/I off = 0.94; lane 10), cannot be sensed effectively in the absence of RNase H. However, in the presence of the latter, the full-match C-allele target gives rise to a significant signal enhancement (I on/I off = 5.7; lane 11), whereas the T-mismatch remains inactive (I on/I off = 1.4;lane 12).

Using “chemically misacylated AMP” which is photogeneratable, tRNA can be enzymatically acylated with N-methylamino acids in an efficiency comparable to that of the corresponding natural aminoacylation process with amino acids.

The “light-up” RNA aptamer–Hoechst pair can be used as a fluorescent tag to monitor transcription processes.