De novo synthesis and development of an RNA enzyme

Ikawa et al. 10.1073/pnas.0405886101.

Supporting Information

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Table 1. Conditions of in vitro selection to yield cis-DSL-01

Table 2. Conditions of in vitro selection from the doped DSL-1 library

Table 3. Activities of cis-DSL-01 and cis-DSL-1

*Calculated final yield was determined by data fitting with a single exponential curve.

Fig. 5. The analysis of the phosphodiester linkage produced at the ligated junction. Cis-DSL-01 was cut at its loop in P2 to produce two fragments for the reconstitution of the ribozyme. The 5' fragment was transcribed in the presence of [a -³²P]GTP to label the 5' phosphate of each guanylate residue. The 5' (5 × 10⁴ cpm) and 3' fragments (5 m M) were assembled together with the substrate (5 m M) and incubated at 37°C for 18 h. The ligated substrate and 5' fragment was purified by electrophoresis on a 10% denaturing polyacryamide gel and digested with RNaseT₂, which does not cleave 2'-5' linkage (1). The products of nuclease digestion were separated by two-dimensional thin-layer chromatography. As a control, an unlabeled 10-mer RNA lacking 5' triphosphate but possessing a 2'-5' linkage between C and A [5'-UCCAC(2'-5')AGGGA-3', purchased from Chem-Genes, Waltham, MA] was used. A circle with fragmented line corresponds to the spot of Cp(2'-5')Ap in the control.

1. Jaeger, L., Wright, M. C. & Joyce, G. F. (1999) Proc. Natl. Acad. Sci. USA 96, 14712-14717.

Fig. 6. (A) Structure and sequence of a pool of DSL-1 variants for the second selection. N in the rectangular box indicates an equivalent mixture of all four bases. Small letters indicate mixed nucleotides composed of the original (61%) together with three other bases (13% each). (B) The consensus sequence of the selected catalytic modular units. Invariable nucleotides are shown with red. Positions where specific base-substitutions resulted in a considerable improvement of the activity are shown with green. n, non-conserved nucleotides; Py , Pyrimidines; X-Y, Watson-Crick or GU-wobble pairs. (C) Sequences and activity of the clones selected from the pool of cis-DSL-1 variants. The selected nucleotides different from those of cis-DSL-1 are shown with red. The nucleotides corresponding to the Ns in A are indicated in the rectangular box.

Table 4. Activities of cis-DSL-1 and cis-DSL-1S with different substrate-P1 sets

ND, not determined.

*Calculated final yield was determined by data fitting with a single exponential curve.

Fig. 7. An autoradiogram of nucleotidyl addition reaction of the variants of cis-DSL-1S. The reaction mixtures were electrophoresed on a 5% denaturing polyacrylamide gel. Asterisks indicate the components labeled with ³²P. The lane at far left shows the reaction of cis-DSL-1S (DSL-1S*) with S-1 substrate (sub1) as a control.

Fig. 8. A time course of the ligation with trans-DSL-2 and the corresponding substrates. 0.1, 1, and 1 m M trans-DSL-2, substrate 1, and substrate 2, respectively, were incubated in the presence of 25, 50, and 30 mM MgCl₂, KCl, and Tris·Cl pH 7.5, respectively, at 37°C.

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