Supplemental Figure 1. Ribose-614 cleavage is unaffected by the addition of 1% complementary strand. Addition of equimolar amounts of the complement greatly lowers cleavage rate and plateau. (A) Cleavage profile for ribose-614 (1 nm)(triangles), or ribose-614 (115 nm) plus complementary strand (11.5 nm)(squares). (B) Cleavage profile for ribose-614 (222 nm)(triangles), or ribose-614 (2 nm) plus equimolar complementary strand (circles). Unless otherwise stated, all kinetics were performed under standard reaction conditions, namely 1M NaCl, 1 mm MgCl 2, 5 mm HEPES, 25 C, and errors in percent cleaved are < ±2 percentage points. Supplemental Figure 2. Cleavage products do not affect ribose-614 cleavage, and only minimally alter ribose-library cleavage. Ribose-614 or ribose-library were incubated either alone (4 nm), or with the addition of the 28-nt product of 614 cleavage (4 nm), the 78-nt product of 614 cleavage (4 nm), or both (4 nm each). Data are fit to a single exponential curve. Errors in percent cleaved are < ±2 percentage points. Supplemental Figure 3. Gel-purification of ribose-614 at cleavage plateau results in additional cleaveage, indicating a fraction of 614 is folded into an inactive conformation. Ribose-614 (222 nm) was allowed to self-cleave for 14, nearing cleavage plateau (triangles, top curve), at which point half the sample was resolved with denaturing PAGE. The fraction of ribose-614 remaining uncleaved at 14 was gel-purified, resuspended in reaction buffer (to a final concentration of 1 nm) and incubated for additional time (diamonds, bottom curve; percentage cleaved as a fraction of the label in the gel purified product, not of initial substrate). Errors in percent cleaved are < ±2 percentage points.
Supplemental Figure 4. Reheating ribose-614 results in additional cleavage, indicating a fraction of ribose-614 is folded into an inactive conformation. Ribose-614 (1 nm) was allowed to react until it reached cleavage plateau (141 h), at which point half of the sample was denatured by heating to 96 C for 3 min, and slowly cooled to 23 C over 1 min. Errors in percent cleaved are < ±2 percentage points. Supplemental Figure 5. Ribose-614 cleavage rate is concentration dependent. (A) Complete time course for ribose-614 at various concentrations. Rates are estimated based on a fit to a single first-order exponential equation. (B) Linear initial phase of time course. Rates are estimated based on the slope of best fit line. Supplemental Figure 6. Cat+ribose competes with ribose-614 for cleavage. Ribose- 614 (1 nm) was incubated with and without cat+ribose (1 nm). Cat+ribose is not catalytic and when incubated alone is not cleaved. Data are fit to a single first-order exponential equation. Supplemental Figure 7. Various substrates can compete with ribose-614 for selfcleavage. A nine fold excess of unlabeled deoxyribose-lib62, deoxyribose-614, deoxyribose-614 C72T, or cat+deoxyribose was added to radiolabeled ribose-614 (33 nm) at time zero. Errors in percent cleaved are < ±2 percentage points. Supplemental Figure 8. The cis-cleavage rate of ribose-614 is not affected by excess competitors. Cleavage assays of ribose-614 (at a low concentration (.33 nm) so as to favor cis-cleavage) were performed with and without the addition of a 9-fold excess of deoxyribose-614 or deoxyribose-lib6lo2. Data are fit to a single first-order exponential equation.
Supplemental Figure 9. Cleavage rates of three different substrates reaches saturation at high concentrations of deoxyribose-614. Substrate saturation experiments were conducted by incubating individual substrates (4 nm) with increasing amounts of deoxyribose-614 enzyme. Various substrates are (A) cat+ribose, (B) ribose-614, and (C) ribose-lib62. Data were fit using a Michaelis-Menten model using Prism software. Initial rates are estimates based on fitting the data to an exponential equation, and therefore correct for cleavage plateaus. Primary data are in Supplemental Fig. 1. Supplemental Figure 1. Primary data used to derive initial rates reported in Supplemental Fig. 9. Cleavage profile for cleavage of cat+ribose (A), ribose-614 (B), and ribose-lib62 (C) by increasing amounts of deoxyribose-614. Data are fit to a single first-order exponential equation. Supplemental Figure 11. Single-turnover kinetics for deoxyribose-614 cleaving cat+ribose (A) or ribose-lib62 (B) at levels of deoxyribose-614 below saturation. Data were fit to first-order exponential curve. Supplemental Figure 12. Compound deoxyribose-614 cleaves substrates with multiple turnover, and cleavage is unaffected by the slow cooling protocol. Substrate (riboselib62 in (A), or cat+ribose in (B)) were incubated in 4-fold excess of deoxyribose-614 (at 33 and 1 nm). The number of turnovers of substrate was calculated by multiplying the percent of substrate cleaved by the initial concentration of substrate and dividing by the concentration of deoxyribose-614. Substrates and deoxyribose-614 were resuspended in reaction buffer, and combined either before (pre-mix) or immediately after (post-mix) heating to 96 C and slowly cooling to 23 C over 1 minutes.
Supplemental Figure 13. The effect of the slow cooling protocol on intra-molecular and inter-molecular ribose-614 cleavage rates. Ribose-614 was incubated at high concentrations (2 nm) favoring trans cleavage (A), and low concentrations (2 nm) favoring cis-cleavage (B), with and without denaturing and folding using the slow cool protocol. Supplemental Figure 14. The number of substrate turnovers remains linear beyond the initial turnover during multi-turnover kinetics of deoxyribose-614 cleaving substrate at various concentrations. Compound deoxyribose-614 (2 nm) was incubated with increasing concentrations of cat+ribose substate. The number of turnovers of cat+ribose was calculated by multiplying the percent of substrate cleaved by the initial concentration of substrate and dividing by the concentration of deoxyribose-614. Supplemental Figure 15. The addition of a chase substrate reduces the residual ribose- 614 cleavage following the chase below that seen under predominately cis-cleavage conditions. The residual cleavage of ribose-614 (2 nm) following the addition of chase (3 mm deoxyribose-614) was calculated by subtracting the time (1 h) and percent cleaved (ca. 3%) at the time the chase was added from the percent cleaved per time point for all subsequent time points. For comparison, data from ribose-614 cleavage under predominately cis-cleaving conditions (1.1 nm) is plotted. Supplemental Figure 16. The cleavage profile of ribose-614 shows that cis-cleavage is unaffected by lowering the incubation temperature. Ribose-614 was incubated at low concentration (2 nm, so as to favor cis-cleavage) at either 4 C or 25 C.
Supplemental Figure 17. The rate of ribose-lib62 trans-cleavage by deoxyribose-614 is increased at lower temperature. Ribose-lib6lo2 substrate (1.5 nm) was incubated with deoxyribose-614 (1 nm) at either 15 C or 25 C.
A 7 6 5 4 3 2 1 5 1 15 2 25 3 B 7 ribose-614 ribose-614 + compl-614 6 5 4 3 2 1 5 1 15 2 25 3 ribose-614 ribose-614 + compl-614 Supplemental Figure 1.
8 7 6 5 4 3 2 1 5 1 15 2 25 3 ribose-614 ribose-614 + 28-nt product ribose-614 + 78-nt product ribose-614 + 28 & 78-nt prod. ribose-library ribose-library + 28-nt product ribose-library + 78-nt product ribose-library + 28 & 78-nt prod. Supplemental Figure 2.
8 7 6 5 4 3 2 1 5 1 15 2 25 3 35 ribose-614 gel purified ribose-614 Supplemental Figure 3.
8 7 6 5 4 3 2 1 5 1 15 2 25 ribose-614 ribose-614 denatured and refolded at 141 Supplemental Figure 4.
Supplemental Figure 5.
6 5 4 3 2 1 5 1 15 2 25 cleavage of ribose- 614 cleavage of ribose- 614 with cat+ribose competitor substrate cleavage of cat+ribose by ribose-614 Supplemental Figure 6.
8 7 6 5 4 3 2 1 Competitor (27 nm) added to ribose-614 d-614 d-614dc72t no competitor d-lib62 cat-ribose 5 1 15 2 25 3 Supplemental Figure 7.
5 25 5 1 15 2 25 3 ribose-614 (.3 nm) ribose-614 (.3 nm) + d-614 (3 nm) ribose-614 (.3 nm) + d-lib62 (3 nm) Supplemental Figure 8.
A.6.5.4.3.2 Substrate = cat+ribose BMAX.56.1 KD 29. R squared.97. 1 2 3 4 5 6 7 [E = d-614] nm B.6.5.4.3.2 Substrate = ribose-614 BMAX.48.1 KD 37.3 R squared.77. 1 2 3 4 5 6 7 [E = d-614] nm C.6.5.4.3.2 Substrate = ribose-lib62 BMAX.49.1 KD 25.5 R squared.61. 1 2 3 4 5 6 7 Supplemental Figure 9. [E = d-614] nm
A 1 75 5 25 5 1 15 2 cat+ribose cleaved by: 25 nm d-614 75 nm d-614 225 nm d-614 675 nm d-614 225 nm d-614 675 nm d-614 B 9 8 7 6 5 4 3 2 1 5 1 15 2 ribose-614 cleaved by: nm d-614 25 nm d-614 75 nm d-614 225 nm d-614 675 nm d-614 225 nm d-614 675 nm d-614 C 7 6 5 4 3 2 1 5 1 15 2 ribose-lib62 cleaved by: 25 nm d-614 75 nm d-614 225 nm d-614 675 nm d-614 225 nm d-614 675 nm d-614 Supplemental Figure 1.
A 7 6 5 4 3 2 1 1 2 3 4 5 6 7 B 8 7 6 5 4 3 2 1 Kexpfit.4213.378.426.269 [cat+ribose] + [d-614] 3 nm + 3 nm 1 nm + 1 nm 3.3 nm + 33 nm 1.1 nm + 11 nm 5 1 15 2 25 3 Kexp fit.21.12.78.4 hour [ribose-lib62] + [d-614] 22.5 nm + 45 nm 7.5 nm + 15 nm 2.5 nm + 5 nm.8 nm + 13 nm Supplemental Figure 11.
A 3 2 1 [ribose-lib62] = 4 nm, pre-mix 4 nm], post-mix 133 nm, pre-mix 133 nm, post-mix B 1 2 3 4 5 4 3 2 1 [cat+ribose] = 4 nm, pre-mix 4 nm, post-mix 133 nm, pre-mix 133 nm, post-mix 1 2 3 4 5 Supplemental Figure 12.
A 75 6 45 3 15 with slow cool without slow cool 1 2 3 4 5 B 5 4 3 2 1 with slow cool without slow cool 5 1 15 2 Supplemental Figure 13.
4 3 d-614 (2 nm) + [cat+ribose] = 1 nm 2 nm 2 3 nm 5 nm 1 1 nm 2 nm 5 1 15 2 Supplemental Figure 14.
25 2 15 1 5 1 2 3 4 5 ribose-614 (1.1 nm) ribose-614 (2 nm) + chase (residual cleavage following chase) Supplemental Figure 15.
5 4 3 2 1 4 C 25 C 5 1 15 2 Supplemental Figure 16.
5 4 3 2 1 15 C 25 C 1 2 3 4 5 hour Supplemental Figure 17.