1. In vitro selection of a novel nuclease-resistant RNA phosphodiesterase
Amber Beaudry, James DeFoe, Shawn Zinnen, Alex Burgin, Leonid Beigelman 
Chemistry & Biology 
Volume 7, Issue 5, Pages (May 2000)
DOI: /S (00)

2. Figure 1
Scheme for 2′-amino-dCTP/dUTP trans selection. Randomized RNA pool with engineered binding arms is allowed to base pair with column-bound substrate. Exposure to magnesium allows for potential cleavage of substrate at unpaired G and dissociation from column.
Chemistry & Biology 2000 7, DOI: ( /S (00) )

3. Figure 2
Progress of initial selection as measured by the cleavage assay. Pools were incubated under single-turnover conditions with labeled substrate and quenched after 4 h. Amount of substrate converted to product is shown. G, generation; reaction conditions: 50 mM Tris–HCl, pH 8.5, 100 mM NaCl, 50 mM KCl, 25 mM MgCl2, 37°C, 10 nM pool, < 1 nM substrate (5′-32P-GCC GUG GGU UGC ACA C-3′).
Chemistry & Biology 2000 7, DOI: ( /S (00) )

4. Figure 3
Progress of optimization under physiological conditions (37°C, 50 mM Tris—HCl, pH 7.5, 140 mM KCl, 10 mM NaCl) as measured by column behavior. Percentage of bound pool eluting from column is shown. As signal rose, additional pressure was exerted on the pool in the form of reduced time of exposure to the magnesium-containing elution buffer.
Chemistry & Biology 2000 7, DOI: ( /S (00) )

5. Figure 4
Secondary structure representation of consensus motif (1-9t), cloned from generation 22. Nucleotides 61–84 were part of the primer-binding site for amplification, a potential site of truncation.
Chemistry & Biology 2000 7, DOI: ( /S (00) )

6. Figure 5
Single-turnover kinetic measurement of the 1-9t motif. Using the Michaelis–Menten equation, kcat = ± min−1 and Km = 8.67 ± 0.89 nM. Reaction conditions: 50 mM Tris-HCl, pH 7.5, 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 5 nM ‘5/12’ substrate (5′-GCCGU G GGUUGCAC ACCU-3′), 37°C.
Chemistry & Biology 2000 7, DOI: ( /S (00) )

7. Figure 6
Magnesium dependence of 1-9t motif (circles), hammerhead 16 (HH16; squares) and chemically stabilized HH16 (diamonds). Conditions for single turnover assay: 50 mM Tris–HCl, pH 7.5, 140 mM KCl, 10 mM NaCl, 100 nM ribozyme, 5 nM substrate, 37°C. ‘5/12’ substrate (5′-GCCGU G GGUUGCAC ACCU-3′) was used for 1-9t. HH16 = 5′-GCG AUG ACC UGA UGA GGC CGA AAG GCC GAA ACG UUC CC-3′; HH16 substrate = 5′-GGG AAC GUC GUC GUC GC-3′; chemically stabilized HH16 = 5′-gscsgs asug acc UGA uGa ggc cga aag gcc Gaa Acg uuc ccB (lower case = 2′-O-methyl nucleotide, upper case = ribonucleotide, U = 2′-C-allyl nucleotide, s = phosphorothioate linkage, B = 3′-3′ inverted abasic).
Chemistry & Biology 2000 7, DOI: ( /S (00) )

8. Figure 7
Dependence on pH of 1-9t motif. Linear curve fit gives a slope of Single turnover kinetics: 50 mM buffer, 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 100 nM 1-9t ribozyme, 5 nM ‘5/12’ substrate (5′-GCCGU G GGUUGCAC ACCU-3′), 37°C. pH 7.0–8.5 with Tris–HCl as buffer; pH 8.92 with CHES as buffer. Activity was also tested at pH 6.0 and 6.5 (with MES as buffer) and there was no detectable cleavage.
Chemistry & Biology 2000 7, DOI: ( /S (00) )

9. Figure 8
Representation of stem truncation and loop replacement analysis. Krel is for the 61-mer 1-9t, measured as described in Table 2.
Chemistry & Biology 2000 7, DOI: ( /S (00) )

10. Figure 9
Serum stability analysis of synthetic 1-9t motif. The assay was performed as described in the Materials and methods section. RNase T1 ladder shows position of G residues.
Chemistry & Biology 2000 7, DOI: ( /S (00) )