Supplementary Information
for
Ligation of RNA oligomers by the Schistosomamansoni Hammerhead Ribozyme in
Frozen Solution
By
Lively Lie,ShwetaBiliya, Fredrik Vannberg, and Roger M. Wartell*
School of Biology, Georgia Institute of Technology, Atlanta Georgia 30332, USA
Parker H. Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology, Atlanta Georgia30332, USA
Content`Page
Figure S1 & Figure S22
Figure S3 & Figure S43
Figure S5 & Figure S64
Figure S75
Figure S86
Thermodynamic Calculation & Figure S97,8
Table S19
Tables S210,11,12
References13
Figure S1Single turnoverreaction of Schist26 HHR cleavage of P1•P2 in frozen NTE solution at -20oC. Solid curved line is the fit of a single exponential equation. The rate constant waskobs,cleavage~ 0.060/min. The plateau value of 0.50 remained unchanged for at least72 hours.
Figure S2 The influence of incubating32P-P1>p in NTE solvent with 10mM MgCl2for different periods of time in the absence of the HHR ribozyme and P2. Samples were removed every 4 hrs, mixed with a stopping solution of 80% deionized formamide and 20 mM EDTA and frozen until all samples were examined by electrophoresis on a denaturing 20% polyacrylamide gel. The first lane on the left shows three markers: 32P-P1 (with no phosphate), 32P-P1>p (2'3' -cyclic phosphate), and 32P-P1p (phosphate on the 3').
Figure S3 Effect of pH of solvent on ligation yield after 24 hr incubation at -20oC. pHvalues of the 50 mM Tris buffered NTE solvent (filled diamonds), which were measured at 25oC,are shown in the plot shifted upward by 1 pH unit (e.g. fraction ligated for pH 7.0 NTE solvent is plotted at pH 8.0).This reflects the estimated influence of temperature on Tris pH as described in text. Data obtained in the 50 mM phosphate buffered solvent are shown as filled squares. In this solvent the sodium chloride concentration was adjusted to maintain 100 mM Na+.
Figure S4 A.Dehydration-induced ligation of 32P-P1•P2;Lane 1 shows32P-P1 as a control. Lane 2 is afreeze-induced ligation in NTE buffer after 24 hours at -20oC. Lane 3 is a 10 μL sample that was centrifuged under vacuum (Speed-Vac) for 2 hours before being quenched with stopping solution. Lane 4 is a sample that was centrifuged under vacuum for 2 hours as in lane 3, and left at 25oC overnight before being quenched. B. Single turnover ligation reaction kinetics of HHRz using 32P-P1>p and P2 carried out at 25 oC in 3.8 M NaCl + 50 mM Tris (8.0) + 1 mM EDTA solution. Reaction procedure is described in Materials and Methods. The solid line is the fit of a single exponential equation yielding a rate constant of kobs,ligation ~1.1 /min
A.B.
Figure S5. Model illustratinghow carboxylate anion (In green) may influence acid-base chemistry to enhance ligation
Figure S6.Plot shows the Jones-Dole ‘B’ coefficients of anions vs. ligation yields for the sodium salts of the anions from Figure 5B. Most of the ‘B’ coefficients were obtained from the review article by Collins (2006). The B coefficients for citrate and thiosulfate were determined from viscosity data of sodium citrate and sodium thiosulfatecompiled by Wolf (1966).The Jones-Dole equation, η/ηo = 1 +Ac½ + Bc,expresses the viscosity η of a solution at 20oC in term of the viscosity of water at 20oC (ηo), the molar concentration of the salt solution, c, and the constants A and B. It can be expressed in another form by defining the function S=(η-ηo)/(ηoc½). Since S = A + Bc½, one can plot S vs c½and determine B from the slope.The data for sulfate and fluoride are indicated with an X. As discussed in the text the sodium salts of these two anions have a much lower solubility than the other salts examined.
Figure S7. A.Plot of relative abundance (reads/106total reads) vs. P2-5N sequences for initial P2-5N library. The P2-5N sequences are numberedaccording to their order of abundance along the x axis.Only the first 200 sequences are given. B.The 10 most abundant sequences are explicitly listed along with their % abundance.
B.
Most abundant sequences % abundance
in initial P2-5N libraryfrom reads/106 reads
CUGGCUUCCACUCUCC9.38
CUGGGUUCCACUGUCC8.12
CUGAGUUCCACUGACC6.34
CUGACUUCCACUGGCC3.96
CUGAGUUCCACUGUCC3.29
CUGAGUUCCACUGGCC2.98
CUGACUUCCACUCUCC2.67
CUGGCUUCCACUGACC1.39
CUGGUUUCCACUGUCC1.34
CUGACUUCCACUGACC1.33
Figure S8
On the left is the Schistosomamansoni hammerhead ribozyme strand (black) and the P1•P2 strand (red). The eight mutant P2 sequences from Table S2 that gave the highest enrichment values are shown from left to right. The enrichment values of the wild type P2 sequence, 0.5, and the mutant P2 sequences are given below each sequence. The blue italicized bases are at the randomized N positions of the initial P2-5N library.
Thermodynamic Calculation onthe Effect of Non-Specific P2 Binding in the Ligation Reaction on the Relative Concentrations ofE•P1•P2 Assemblies.
Thermodynamic calculations were made to determineifplausible differences in the affinity of individual P2 sequences to non-specific targetscan affect therelative concentrations of E•P1•P2 assemblies.That is,cannon-specific bindingsequester some P2 sequencesmore than others reducing their availability for ligation? This would affect the concentrations of ligatable assembliesin a manner unrelated to the fitness of P2 sequences for ligation.
The model used for the calculationassumed four species; E, P1, P2, and P2N, where P2N is a pseudo specie representing the average of all P2-5N oligomers other than P2. Eleven coupled equilibrium reactions describe the formation of thecomplexes E•P1•P2, E•P1, E•P2, P2•P1, P2•P2, P1•P1, E•E, E•P2N, P1•P2N, P2•P2N, and E•P1•P2N. The program HySS2009 was used to solve the coupled equations and evaluate equilibrium concentrations of the complexes (Alderighi et al. 1999).
The free energies of the reactionsthat form the various complexes were calculated using the RNAup algorithm (Muckstein et al. 2006; Lorenz et al. 2011) with the parameter set of Turner and Mathews (Turner and Mathews 2010). RNAup calculates binding free energies by considering the free energy requiredto open segments of secondary structuresof theinteracting RNAs as well as the freeenergy gained from duplex formation.For complexes involving specie X (with X=P1, P2 or E) and the P2N pseudo specie, the binding free energy was the average free energy of binding X to the 100 most abundantP2-5N oligomers in the initial library. An acknowledged limitation to this calculation is that the empirical free energy parameters were obtained in a solvent of 1 M Na+, and they may not be appropriate for the 3 to 4 M Na+ eutectic conditions.
We assumedtotal concentrations of [E]t, [P2-5N]t, [P1>p]t based onthe ligation reaction used to preparethe ligated P2 library.The room temperature concentration values were increased 30 fold to simulate the effect that freezinghas on the concentration of solutes in the interstitial liquid(Franks and Royal Society of Chemistry (Great Britain) 2000). Thus, [E]t = (30 x 5 μM) or 150 μM, [P2-5N]t=(30 x 25 μM) or 750 μM, and [P1>p]t= (30 x 1 μM) or 30 μM. The total concentration of an individual P2 sequence was the product of its fractional abundance in the initial P2-5N library (Table S2) times[P2-5N]t.
Table S2 lists free energies (ΔGo) for the formation of the E•P2 complexes. As expected, the wild type P2 has the lowest free energy.The free energies of complexes betweenmutant P2’s and the E strand are less favorableand depend on the number and nature of their mismatches.
Figure S9 (shown below) shows ΔGovalues for binding wild type P2 (▪) and P2-GG-UGU (○) to the 100 most abundant sequences of the initial P2-5N library. The P2-5N sequences are numbered along the x axis in their order of abundance with 1 the most abundant. As might be expected ΔGo values vary depending on the specific pair of P2 sequences involved. Visual inspection of Figure S9suggests that wild type P2 has, on average, a lower free energy and thus greater affinity than P2-GG-UGU to the ensemble of P2-5N sequences. Indeed, calculation of the average ΔGo valuesyielded <ΔGo = -6.58 kcal/mole for binding wild type P2 tothe P2-5N species,and <ΔGo = -4.17 kcal/mole for binding P2-GG-UGU to the P2-5N species. Wild type P2 has a greateraverage affinity than P2-GG-UGU for the P2N pseudo specie.
When the equilibrium concentrations of E•P1•P2 were calculated for wild type P2 (wtP2) and P2-GG-UGU using the above parameters and concentrations, we found [E•P1•P2-GG-UGU] =1.62 μM and [E•P1•wtP2] = 0.76 μM. However if we assume the non-specific binding of P2-GG-UGU to the P2N pseudo specie is the same as wtP2, both with an interaction free energy of ΔGo = -6.58 kcal/mol , then [E•P1•P2-GG-UGU] = 0.24μM. The concentration of E•P1•wtP2 assemblies is now three-fold greater than [E•P1•P2-GG-UGU]. This result illustrates that plausible differences in non-specific binding of P2 sequences to the pool of P2-5N oligomers can influence the relative concentrations of E•P1•P2 assemblies. The relative abundance and apparent enrichment values of ligated P2 sequences may reflect factors in addition to their fitness for ligation.
Figure S9
TABLE S1
Influence of soluteadditions on freezing-induced ligation.Effect of various solutes added to NTE solvent(100 mMNaCl +50 mM Tris (pH 8.0) + 0.1 mM EDTA) on freezing-induced ligation of P1>p and P2 by HHR at -7oC (or -20oC if indicated). Yields are average of three experiments and are ± 2%.Ligation reactions conducted are described in Materials and Methods.
Condition% ligation yield after 24 hrs
NTE at -7oC30
Proline 27
Threonine 24
Glutamine 25
Cysteine 20
Alanine 27
Valine 21
Lysine 29
Arginine 21
Methionine 28
Glutamic Acid 20
Histidine 24
Leucine 26
Glycine 28
Isoleucine 7
Asparagine 26
Serine 27
Phenylalanine 28
Tyrosine 29
Tryptophan 4
Aspartic Acid 28
10 mMadded
50mM added
Glucose22
Lactose30
Sucrose22
Trelhalose26
1% glycerol25
100 mM TMAO 28 10 mM spermine* 14
10 mM spermidine25
*- an additional slow mobility band was observed
representing 12% of the total P1
Table S2
Results from ligation of P1 to P2-5N library by hammerhead ribozyme in frozen solution. Ligated P2 sequences are listed based on reads/106 reads from HTS (column 3). The positions in the 16 nt P2 sequences that were randomized (N) in the P2-5N library are highlighted in yellow. Reads/106 reads of theP2 sequences in the initial P2-5N library were determined from HTS (column 5). The free energy, ΔGo, of binding each P2 sequence to the E strand was determined using the RNAup algorithm as described above. Fold enrichment is col. 3 number divided by col. 5 number.
Reads/106 / fold enrichment / reads/106 / ΔGo ofof ligated / from initial / in initial / P2•E
Rank / Ligated P2 Sequences / P2 sequences / P2-5N library / P2-5N library / (kcal/mol)
1 / GG / UUCCAC / UGU / 364713 / 4.5 / 81172 / -5.32
GG / UUCCAC / UGG / 170857 / 15.3 / 11141 / -8.24
GG / UUCCAC / UGA / 61995 / 7.1 / 8734 / -3.67
AG / UUCCAC / UGU / 44902 / 1.4 / 32894 / -2.99
GG / UUCCAC / GUU / 38531 / 9.3 / 4144 / -5.44
UG / UUCCAC / UGU / 36784 / 4.8 / 7654 / -3.09
UA / UUCCAC / UGU / 20199 / 19.9 / 1015 / -3.14
GU / UUCCAC / UGU / 17828 / 1.3 / 13448 / -3.05
UU / UUCCAC / UGU / 17552 / 3.9 / 4514 / -3.15
10 / AG / UUCCAC / UGG / 13461 / 0.5 / 29795 / -4.35
GG / UUCCAC / UUG / 11252 / 1.6 / 7038 / -10.09
GG / UUCCAC / GUA / 10500 / 5.9 / 1779 / -6.94
GA / UUCCAC / UGG / 8919 / 4.9 / 1820 / -9.77
GG / UUCCAC / UUU / 8410 / 1.1 / 7361 / -4.64
GG / UUCCAC / GCG / 7429 / 5.8 / 1291 / -8.03
GA / UUCCAC / UGU / 7141 / 1.6 / 4338 / -3.74
AA / UUCCAC / UGU / 6834 / 6.1 / 1127 / -3.14
AG / UUCCAC / UGA / 6479 / 0.1 / 63399 / -2.12
GU / UUCCAC / UGG / 6429 / 0.6 / 10912 / -5.85
20 / GG / UUCCAC / GUG / 5881 / 1.4 / 4203 / -6.84
GU / UUCCAC / GUU / 5302 / 3.9 / 1374 / -3.36
GG / UUCCAC / GCU / 5276 / 4.6 / 1156 / -7.14
UU / UUCCAC / UGG / 4979 / 0.9 / 5406 / -4.05
GG / UUCCAC / GGU / 4759 / 2.0 / 2407 / -5.13
GG / UUCCAC / UCG / 4740 / 2.8 / 1702 / -11.31
GG / UUCCAC / UGC / 4428 / 1.2 / 3751 / -3.87
GU / UUCCAC / UGA / 3980 / 1.4 / 2876 / -1.1
GG / UUCCAC / GCA / 3736 / 6.7 / 558 / -7.01
GU / UUCCAC / UCG / 3596 / 1.1 / 3375 / -10.35
30 / UU / UUCCAC / UGA / 3586 / 1.8 / 1990 / -2.11
GU / UUCCAC / UUU / 3527 / 1.6 / 2207 / -2.92
UG / UUCCAC / UGG / 3083 / 0.4 / 7449 / -5.43
GG / UUCCAC / CUG / 3052 / 7.6 / 399 / -7.99
GG / UUCCAC / UCU / 2854 / 0.8 / 3575 / -6.06
GA / UUCCAC / GCG / 2799 / 12.2 / 229 / -9.19
AA / UUCCAC / UGG / 2799 / 0.5 / 6105 / -4.19
GG / UUCCAC / UAG / 2784 / 1.9 / 1503 / -5.08
AA / UUCCAC / UGA / 2624 / 7.6 / 346 / -2.26
UA / UUCCAC / UGG / 2474 / 1.3 / 1937 / -5.7
40 / AU / UUCCAC / UGA / 2311 / 0.9 / 2612 / -2.87
UA / UUCCAC / UUU / 2265 / 13.8 / 164 / -1.77
GG / UUCCAC / CUU / 2060 / 7.2 / 288 / -5.5
AU / UUCCAC / UGU / 2028 / 0.7 / 2718 / -2.94
GA / UUCCAC / GUU / 2001 / 6.2 / 323 / -5.32
GG / UUCCAC / GUC / 1974 / 1.8 / 1098 / -6.57
GG / UUCCAC / CGU / 1958 / 3.3 / 593 / -5.27
GA / UUCCAC / UGA / 1933 / 0.9 / 2225 / -5.08
AU / UUCCAC / UGG / 1792 / 0.2 / 8405 / -4.17
GU / UUCCAC / GCU / 1745 / 1.4 / 1209 / -5.69
50 / GU / UUCCAC / UUG / 1657 / 0.3 / 4907 / -7.83
UG / UUCCAC / UGA / 1622 / 0.1 / 11293 / -1.96
GG / UUCCAC / UUA / 1575 / 0.5 / 2988 / -4.22
GG / UUCCAC / GGG / 1469 / 0.8 / 1931 / -5.08
AG / UUCCAC / GUU / 1409 / 0.7 / 1931 / -2.8
GU / UUCCAC / GCG / 1391 / 0.7 / 2072 / -6.11
GG / UUCCAC / UAU / 1385 / 1.0 / 1420 / -3.26
GA / UUCCAC / GUA / 1383 / 10.2 / 135 / -7.92
AA / UUCCAC / GUA / 1381 / 6.5 / 211 / -4.23
GU / UUCCAC / UGC / 1353 / 1.2 / 1162 / -4.69
60 / GA / UUCCAC / UUG / 1308 / 2.0 / 652 / -11.28
AA / UUCCAC / UUU / 1245 / 7.9 / 158 / -1.26
GA / UUCCAC / GCA / 1201 / 25.6 / 47 / -8.33
GU / UUCCAC / GUA / 1142 / 1.7 / 687 / -3.65
AA / UUCCAC / GCA / 1135 / 10.7 / 106 / -2.7
GG / UUCCAC / CGG / 1127 / 1.7 / 646 / -4.64
AA / UUCCAC / UUG / 1106 / 1.1 / 963 / -5.54
AA / UUCCAC / UUA / 1068 / 13.0 / 82 / -1.1
GA / UUCCAC / GCU / 1061 / 0.4 / 2360 / -8.28
UG / UUCCAC / GUU / 1060 / 2.3 / 458 / -3.36
70 / AA / UUCCAC / GCG / 1047 / 4.0 / 264 / -3.93
AU / UUCCAC / GUU / 1042 / 2.3 / 452 / -2.86
AA / UUCCAC / GUU / 1042 / 6.3 / 164 / -2.46
GU / UUCCAC / GCA / 1019 / 4.3 / 235 / -5.24
GU / UUCCAC / GUC / 989 / 1.8 / 552 / -5.4
GG / UUCCAC / ACG / 969 / 4.6 / 211 / -8.17
GU / UUCCAC / UUA / 966 / 1.1 / 892 / -2.16
GG / UUCCAC / UUC / 921 / 0.4 / 2095 / -5.2
GU / UUCCAC / GUG / 915 / 0.1 / 6257 / -3.97
AG / UUCCAC / UUG / 884 / 0.2 / 3839 / -5.22
80 / UG / UUCCAC / UUU / 857 / 1.0 / 880 / -2.02
AA / UUCCAC / UUC / 853 / 8.1 / 106 / -2.61
AG / UUCCAC / UUU / 849 / 0.4 / 2131 / -1.9
GG / UUCCAC / AGU / 834 / 3.0 / 276 / -4.95
AA / UUCCAC / GUC / 823 / 5.4 / 153 / -4.72
UA / UUCCAC / UCA / 789 / 7.9 / 100 / -3.76
UA / UUCCAC / GUU / 787 / 13.4 / 59 / -2.82
AA / UUCCAC / GAA / 785 / 4.3 / 182 / -1.06
GU / UUCCAC / UCU / 784 / 0.1 / 6639 / -4.86
UA / UUCCAC / UGA / 758 / 5.2 / 147 / -2.24
90 / GA / UUCCAC / UCG / 752 / 0.5 / 1444 / -13.33
GG / UUCCAC / AUG / 737 / 0.6 / 1168 / -6.29
UA / UUCCAC / UCU / 697 / 0.8 / 822 / -3.38
UU / UUCCAC / UCU / 685 / 0.5 / 1450 / -2.72
GC / UUCCAC / UGU / 676 / 0.1 / 8364 / -2.55
GA / UUCCAC / UGC / 673 / 2.0 / 340 / -8.4
AG / UUCCAC / UGC / 651 / 0.2 / 3939 / -4.53
GG / UUCCAC / GGA / 616 / 0.7 / 880 / -2.23
AG / UUCCAC / CUG / 611 / 0.4 / 1726 / -5.32
GA / UUCCAC / GUG / 611 / 1.3 / 470 / -7.91
100 / GU / UUCCAC / CUU / 599 / 6.4 / 94 / -4.23
References
Alderighi L, Gans P, Ienco A, Peters D, Sabatini A, Vacca A. 1999. Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species. Coordin Chem Rev184: 311-318.
Franks F, Royal Society of Chemistry (Great Britain). 2000. Water a matrix of life. In RSC paperbacks, pp. xii, 225 p. Royal Society of Chemistry,, Cambridge, UK.
Lorenz R, Bernhart SH, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL. 2011. ViennaRNA Package 2.0. Algorithms for molecular biology : AMB6: 26.
Muckstein U, Tafer H, Hackermuller J, Bernhart SH, Stadler PF, Hofacker IL. 2006. Thermodynamics of RNA-RNA binding. Bioinformatics22: 1177-1182.
Turner DH, Mathews DH. 2010. NNDB: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure. Nucleic Acids Res38: D280-282.
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