pubs.acs.org/Biochemistry Published on Web 09/15/2010 r 2010 American Chemical Society

9058 Biochemistry 2010, 49, 9058–9062

DOI: 10.1021/bi101164s

Thermodynamic Characterization of RNA Triloops†

Praneetha Thulasi, Lopa K. Pandya, and Brent M. Znosko*

Department of Chemistry, Saint Louis University, Saint Louis, Missouri 63103, United States

Received July 21, 2010; Revised Manuscript Received September 14, 2010

ABSTRACT: Relatively few thermodynamic parameters are available for RNA triloops. Therefore, 24 stem-loop sequences containing naturally occurring triloops were optically melted, and the thermodynamicparameters ΔH�, ΔS�, ΔG�37, and TM for each stem-loop were determined. These new experimental values,on average, are 0.5 kcal/mol different from the values predicted for these triloops using themodel proposed byMathews et al. [Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H.(2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7287-7292]. The data for the 24 triloops reported here were thencombined with the data for five triloops that were published previously. A new model was derived to predictthe free energy contribution of previously unmeasured triloops. The average absolute difference between themeasured values and the values predicted using this proposed model is 0.3 kcal/mol. These new experimentaldata and updated predictive model allow for more accurate calculations of the free energy of RNA stem-loops containing triloops and, furthermore, should allow for improved prediction of secondary structure fromsequence.

RNA stem-loops containing three nucleotides in the loop,triloops, are common secondary structuremotifs found in naturallyoccurring RNA. For example, bacterial 16S rRNAs strongly favortetraloops; however, theUUUtriloop is themost common replace-ment (1). In the 16S-like rRNA variable regions, triloops accountfor 7% of the loops in bacteria and 16%of the loops in eukaryotes(2). Triloops are also found in large subunit rRNAs (3, 4), 5SrRNAs (5), signal recognitionparticles (6),RNasePRNAs (7), andgroup I introns (8, 9). More specifically, triloops are found inBrome mosaic virus (þ) strand RNA (10), human rhinovirusisotype 14 (11), iron responsive element RNA (12), and an RNAaptamer for bacteriophage MS2 coat protein (13), to name a few.Although relatively unstable due to the strain in the loop, triloopsmay be an important structural feature due to the accessibility ofthe loop nucleotides for recognition by proteins, other nucleicacids, or smallmolecules. It has been shown that triloops play a rolein various biological processes, including virus replication (11, 14),viral synthesis (15), and iron response (12), to name a few.Nevertheless, only a few studies have thermodynamically charac-terized RNA triloops (16-20), and only three of these (16-18)were done using 1 M NaCl (the salt concentration in which mostnearest neighbor parameters are derived).

The current model used by secondary structure predictionalgorithms to predict the thermodynamic contribution of RNAtriloops to stem-loop stability is sequence independent; alltriloops contribute 5.4 kcal/mol to stem-loop stability, withthe exception of 50CCC30 which contributes 6.9 kcal/mol (21). Inaddition, there are two unstable triloop sequences (50CAACG30

and 50GUUAC30) for which this predictive model is not used;

instead, the ΔG�37,loop values (6.8 and 6.9 kcal/mol, respectively)for these two triloops are provided in a lookup table (21). Aninteresting study by the Bevilacqua laboratory (19) used acombinatorial approach and temperature gradient gel electro-phoresis to identify stable and unstable RNA triloops. It wasdiscovered that sequence preferences for exceptionally stabletriloops included a U-rich loop and C-G as the closing base pair.Although they used 10 mM NaCl during their melting experi-ments, they suggested that the rules for predicting triloop stabilityat 1MNaCl should bemodified; however, this has yet to be done.Here, we report the thermodynamic parameters for 24 previouslyunmeasured RNA triloops in 1MNaCl and propose a new algo-rithm for predicting the contribution of triloops to stem-loopstability, which includes two bonuses for stabilizing sequencefeatures.

MATERIALS AND METHODS

Compiling and Searching a Database for RNA Triloops.The initial aim of this project was to identify the most frequentlyoccurring RNA triloops in nature and to thermodynamicallycharacterize these hairpin triloop sequences. Therefore, a data-base of 1349 RNA secondary structures containing 123 smallsubunit rRNAs (22), 223 large subunit rRNAs (3, 4), 309 5SrRNAs (5), 484 tRNAs (23), 91 signal recognition particles (6), 16RNase P RNAs (7), 100 group I introns (8, 9), and 3 group IIintrons (24) was compiled. This database was searched fortriloops, and the number of occurrences for each type of triloopwas tabulated. In this work, G-U pairs are considered to becanonical base pairs.Design of Sequences for Optical Melting Studies. Since

most thermodynamic parameters for RNA secondary structuremotifs are reported for RNA solutions containing 1MNaCl, themelting buffer used in this work also contained 1 M NaCl. Amajor limitation of a thermodynamic analysis of RNA hairpinsusing this high salt concentration is the possible bimolecular

†This work was supported by Award Number R15GM085699 fromtheNational Institute of GeneralMedical Sciences. The content is solelythe responsibility of the authors and does not necessarily represent theofficial view of theNational Institute of GeneralMedical Sciences of theNational Institutes of Health.*To whom correspondence should be addressed. Phone: (314) 977-

8567. Fax: (314) 977-2521. E-mail: znoskob@slu.edu.

Article Biochemistry, Vol. 49, No. 42, 2010 9059

association ofRNA strands (19, 25). To ensure that unimoleculartriloop formation out-competed bimolecular association in a 1MNaCl solution, the following equations, derived from the equi-librium equations and ΔG� = -RT ln K, were utilized:

½H� ¼ - 1þ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þðð8KD½A�TÞ=ðKHKHÞ

p Þð4KDÞ=ðKHKHÞ ð1Þ

½D� ¼ ð½A�T - ½H�Þ=2 ð2Þ

% H ¼ ½H�=ð½H� þ ½D�Þ � 100 ð3ÞHere, [H] is the concentration of hairpin, KD is the equilibriumconstant for duplex formation, [A]T is the total strand concen-tration, KH is the equilibrium constant for hairpin formation,[D] is the concentration of duplex, and%H is the percent hairpinin solution.1 KH and KD values were calculated at 37 �C usingΔG�37 values from RNAstructure (21, 26, 27) for hairpin andduplex formation, respectively. Calculations were done for[A]T = 1 μM and 0.1 mM, which are the typical concentrationrange for the melting experiments. Due to potential competitionfrom duplex formation, many of the most frequently occurringtriloops were not studied here; only those that were likely to formtriloops were used. All of the sequences studied here had% [H]>84% at [A]T = 0.1 mM and % [H] > 99% at [A]T = 1 μM.

Sequences of triloops and closing base pairs were designed torepresent those found in the database described above. Each stemcontained three Watson-Crick pairs in addition to the closingbase pair. The terminal base pair was always a G-C pair in orderto prevent end fraying of the sequence during melting. Care wastaken to design the stem-loop sequences so that the triloop ofinterest would form, with little competition fromother secondarystructure motifs.RNA Synthesis and Purification. Oligonucleotides were

ordered from Integrated DNA Technologies (Coralville, IA). Thepurification of the oligonucleotides followed standard proceduresthat were described previously (28).Optical Melting Experiments and Thermodynamics.

Optical melting experiments were performed in 1 M NaCl,

20 mM sodium cacodylate, and 0.5 mM Na2EDTA (pH 7.0).All stem-loopsweremelted about nine timeswith approximatelya 50-fold concentration range. Each stem-loop melting curveresulted in a single transition, and all melts of a given stem-loopwere concentration independent, suggesting stem-loop forma-tion. Stem-loop thermodynamics were determined by averagingthe thermodynamics derived from each individual curve fit. Thethermodynamic contributions of triloops to stem-loop thermo-dynamics (ΔG�37,triloop, ΔH�triloop, and ΔS�triloop) were deter-mined by subtracting theWatson-Crick contribution (29) from themeasured thermodynamics. Stem sequences in which the terminalpair of the stem (or the triloop closing base pair) is A-U,U-A,G-U,or U-G utilize the 0.45 kcal/mol terminal A-U penalty (29) whencalculating the contribution of the Watson-Crick stem; thus, this0.45 kcal/mol contribution is not included in the parameters forpredicting the loop contribution to stem-loop stability.Linear Regression and Triloop Thermodynamic Para-

meters. Data collected for the 24 triloops in this study werecombined with previously published data for five triloops(16-18) that were also melted in 1 M NaCl. Data for seventriloops measured in these previous studies (16-18) were omittedfrom the analysis and discussion here because, using eqs 1-3,they resulted in%H<75%. Additional thermodynamic data areavailable for triloops in 10 mM sodium phosphate (17, 19, 20)and 0.1 M NaCl (16) but, due to different salt concentrations,were not included in the analysis described here.

A predictive model was derived by linear regression as describedpreviously (28). The calculated experimental contribution of thetriloop to stem-loop stability was used as a constant when doinglinear regression. Many combinations of variables were tested. Tosimultaneously solve for each variable, the LINEST function ofMicrosoft Excel was used for linear regression.

RESULTS

Database Searching. A database of RNA secondary struc-tures was searched for triloops. In this database, 822 triloopswerefound, averaging about one triloop for every two secondarystructures. Table 1 shows a summary of the database resultsobtained. Supporting Information Table S1 shows the completeresults. The first set of data in Table 1 lists frequency and percentoccurrence when the triloop nucleotides and the closing base pairare specified. Because the stability of hairpin loops depends onboth the identity of the nucleotides in the loop and the closing

Table 1: Summary of Database Search Results for RNA Triloopsa

data set 1: triloop with

adjacent base pair data set 2: triloop

data set 3: adjacent

base pairs

data set 4: triloop nucleotides classified as

purine (R) or pyrimidine (Y)

triloopa freqb %c triloopa freqb %c closing bp freqb %c triloopa freqb %c

GGGGC 106 12.9 GGG 108 13.1 C-G 345 42.0 RRR 207 25.2

CGCAG 53 6.4 UAA 96 11.7 G-C 219 26.6 RYR 181 22.0

CUAAG 45 5.5 GCA 66 8.0 U-A 163 19.8 YRR 123 15.0

UUAAA 41 5.0 UUU 53 6.4 A-U 56 6.8 YYY 120 14.6

CAUAG 34 4.1 AUA 48 5.8 U-G 26 3.2 YYR 53 6.4

UGAAA 24 2.9 AAA 32 3.9 G-U 13 1.6 YRY 47 5.7

CUUUG 24 2.9 GAA 32 3.9 RYY 45 5.5

CUCUG 17 2.1 GUA 32 3.9 RRY 41 5.0

CAGAG 15 1.8 UCU 24 2.9

GGUAC 15 1.8 AGA 22 2.7

aNot all combinations in data sets 1 and 2 are shown due to space limitations. For each set of sequences, the strand is writen 50 to 30. bFrequency ofoccurrence in the secondary structure database described in Materials and Methods. cPercent out of 822 triloops, the total number of triloops found in thesecondary structure database.

1Abbreviations: [A]T, the total strand concentration; [D], concentra-tion of duplex; [H], concentration of hairpin; KD, equilibrium constantfor duplex formation; KH, equilibrium constant for hairpin formation;R, purines; Y, pyrimidines; % H, percent hairpin in solution.

9060 Biochemistry, Vol. 49, No. 42, 2010 Thulasi et al.

base pair (16-18, 30-38), this categorization is most important.Categorizing triloops in this fashion results in 177 types oftriloops in the database. The 10 triloop types listed in the firstdata set (Table 1) account for 45%of the total number of triloopsfound. The 167 types of triloops not shown account for theremaining 55%; however, each type represents <1.8% of thetotal number of triloops found.

The second set of data (Table 1) lists frequency and percentoccurrence when only the triloop sequence is specified (the closingbase pair is not considered). Categorizing triloops in this fashionresults in 62 types of triloops in the database; however, there are64 sequence possibilities. Therefore, two sequence possibilities,50AGG30 and 50CUG30, are not found in the database. The 10triloops listed in the second data set (Table 1) account for 62% ofthe total number of triloops found. The 52 types of triloops notshown account for the remaining 38%, with each triloop repre-senting <2.5% of the total number of triloops found. If theoccurrence of triloops were completely random, we would expecteach triloop sequence to occur ∼13 times in the database.

The third set of data (Table 1) lists frequency and percentoccurrence of the closing base pair. Categorizing triloops in thisfashion results in six types of closing base pairs in the database,representing all possible types. If the occurrence of triloops werecompletely random, we would expect each closing base pair tooccur 137 times in the database.

The fourth set of data (Table 1) lists frequency and percentoccurrence of the triloop nucleotides when A and G are categor-ized as purines (R) and C and U are categorized as pyrimidines(Y). Categorizing triloops in this fashion results in eight types oftriloops, representing all possible combinations. If the occurrenceof triloops were completely random, we would expect each type tooccur ∼100 times in the database.Thermodynamic Parameters. As discussed earlier, due to

possible bimolecular association of strands, many of the mostfrequently occurring triloops were not studied. All of the triloopsstudied here were found in the secondary structure databasedescribed above andweremost likely in the hairpin conformationduring the optical melting studies (as determined by usingeqs 1-3). Table 2 shows the thermodynamic parameters ofhairpin formation that were obtained from the average of fittingeach melting curve to the two-state model. Data for 29 stem-loops containing unique triloops are shown in order of decreasingfrequency as determined by the database search described above.Interestingly, all five of the triloops studied previously are of thesequence 50ANA30.Contribution of Triloops to Stem-Loop Free Energy.

The contributions of the 29 triloops to stem-loop stability arealso listed in Table 2. The examination of the free energycontributions of triloops to stem-loop free energy indicates alarge variance, withΔG�37,triloop ranging from 4.5 to 6.8 kcal/mol.

Table 2: Thermodynamic Parameters for Stem-Loop Formation and Contributions of Triloops to Stem-Loop Stabilitya

frequencyb sequencecΔH�

(kcal/mol)

ΔS�(eu)

ΔG�37(kcal/mol)

TMd

(�C)ΔH�triloope

(kcal/mol)

ΔG�37,triloope

(kcal/mol)

predicted

ΔG�37,triloopf

(kcal/mol)

34 GGCAUAGCCg -21.9( 1.9 -67.5( 6.0 -0.94( 0.15 51.0 6.4 5.74 5.2

15 GGCCAGAGGCC -39.8( 2.1 -113.9( 6.2 -4.46( 0.22 76.2 1.9 5.48 5.2

14 GCCUUUUAGGC -36.2( 1.8 -105.6( 5.4 -3.45( 0.15 69.7 2.6 4.86 4.7

12 GGCUAAAAGCC -29.9( 2.5 -88.5( 7.8 -2.47( 0.18 64.9 8.9 5.84 5.9

10 GGGAUACAAAGUAUCCAh -56.2 -160.9 -6.3 76.4 3.7 5.38 5.2

10 GGCCUUCGGCC -36.1( 2.8 -102.1( 7.9 -4.38( 0.36 79.9 5.6 5.56 5.2

9 GCCGUUUCGGC -43.4( 3.5 -125.7( 10.7 -4.40( 0.17 72.0 -4.5 4.64 4.7

8 GGCGACACGCC -29.1( 3.1 -85.4( 9.7 -2.66( 0.22 68.2 9.8 6.38 5.9

8 GGCUCAAAGCC -27.9( 2.7 -82.2( 8.5 -2.40( 0.12 66.1 10.9 5.91 5.9

7 GGCAUAUUGCC -33.6( 1.4 -99.1( 4.5 -2.85 ( 0.06 65.7 5.1 5.49 5.9

7 GGCUUAUAGCC -31.7( 1.6 -92.9( 4.7 -2.85( 0.14 67.7 7.1 5.46 5.9

7 GGCCUCCGGCC -39.4( 2.5 -110.6( 7.3 -4.77( 0.19 80.1 2.3 5.17 5.2

6 GGCGAGACGCC -37.0( 2.5 -109.3( 8.0 -3.11( 0.25 65.4 1.9 5.93 5.9

6 GGGAUACCCg -27.6( 1.7 -88.3( 5.1 -0.26( 0.22 38.9 -0.8 6.26 5.9

6 GGCGCUUCGCC -26.7( 2.8 -78.7( 8.8 -2.28 ( 0.12 66.0 12.2 6.76 5.9

5 GGCUACAAGCC -30.7( 1.9 -90.7( 6.0 -2.61( 0.15 65.7 8.1 5.70 5.9

5 GCCCGAAGGGC -43.4( 1.9 -122.7( 5.8 -5.35( 0.07 80.6 -1.7 4.59 5.2

5 GCCCUAUGGGC -42.6( 1.9 -120.4( 6.8 -5.24( 0.17 80.6 -0.9 4.70 5.2

4 GGCAAAAUGCC -30.0( 2.2 -89.2( 6.6 -2.36( 0.17 63.5 8.7 5.98 5.9

4 GGCGAAACGCC -38.0( 2.3 -110.9( 7.3 -3.63 ( 0.13 69.8 0.9 5.41 5.9

4 GGCCACAGGCC -38.2( 1.7 -109.8( 5.0 -4.16( 0.17 74.9 3.5 5.78 5.2

4 GGCGCAACGCC -33.0( 1.9 -95.4( 5.7 -3.42( 0.15 72.9 5.9 5.62 5.9

4 GGCACAUUGCC -31.6( 2.4 -93.8( 7.4 -2.47( 0.16 63.3 7.1 5.87 5.9

4 GGCCCUUGGCC -51.8( 1.6 -149.3( 4.8 -5.47 ( 0.22 73.6 -10.1 4.47 5.2

2 GGCUAACGGCC -34.5( 1.4 -101.8( 4.4 -2.91( 0.08 65.6 5.9 5.40 5.9

2 GGCGACCCGCC -35.3( 1.2 -103.4( 3.6 -3.23( 0.19 68.3 3.6 5.81 5.9

1 GGCUACCAGCC -30.9( 2.3 -91.1( 7.3 -2.63( 0.10 65.9 7.9 5.68 5.9

0 GGGAAAUCCi -18.4( 2.9 -60.4( 9.2 0.3( 0.2 31.8 3.3 6.37 5.9

0 GGAGAAAUUCCi -31.2( 3.6 -97.6( 11.9 -0.9( 0.4 46.1 -2.2 6.34 5.9

aMeasurements were made in 1.0 M NaCl, 10 mM sodium cacodylate, and 0.5 mM Na2EDTA at pH 7.0. bFrequency of occurrence in the databasedescribed inMaterials andMethods. cThe sequences are written 50 to 30. The nucleotides in the triloop are underlined for easy identification. dThe averageTM

difference between the most and least concentrated sample for each stem-loop was 2.7 �C. If the sequences were forming a duplex, nearest neighborthermodynamic values (29) predict that the TM should change by about 16 �C for the typical concentration range used here, suggesting the formation ofstem-loop structures. eThe enthalpy and free energy contribution of the triloop were calculated by subtracting the Watson-Crick contribution of thestem (29) from the experimental enthalpy and free energy of the stem-loop, respectively. fPredicted triloop free energy contributions using the new predictivemodel shown in eq 4. gReference 16. hReference 17. iReference 18.

Article Biochemistry, Vol. 49, No. 42, 2010 9061

Interestingly, the 50CUU30 triloop is both the least and moststable triloop reported here, depending on its adjacent pair. Itis the least stable triloop (ΔG�37,triloop = 6.8 kcal/mol) whenadjacent to aG-C pair and themost stable triloop (ΔG�37,triloop=4.5 kcal/mol) when adjacent to a C-G pair.Updated Model for Predicting the Free Energy of Pre-

viously Unmeasured Triloops. Currently, in order to predictthe free energy contribution of triloops, all triloops are assigneda free energy penalty of 5.4 kcal/mol. The only modificationto this penalty is for a triloop containing all C residues, whichincludes an additional 1.5 kcal/mol penalty (21) and a lookuptable for two unstable triloops, 50CAACG30 (6.8 kcal/mol) and50GUUAC30 (6.9 kcal/mol) (21).When predicting the free energycontributions of the 29 triloops reported here, experimentalvalues can now be used. For triloops that still do not haveexperimental values, a predictive model can be utilized.

The current predictive model was applied to all of the triloopsreported here. On average, the predicted value was 0.5( 0.4 kcal/mol different than the experimental value. The average thermo-dynamic contribution of the triloops reported here was 5.6 kcal/mol. Using this average value in place of the 5.4 kcal/molpenalty (21) results in an average difference between the predictedvalue and the experimental value of 0.4 ( 0.4 kcal/mol.

In an attempt to further improve prediction, various othermodels were tried. The best model (lowest difference betweenexperimental and predicted values with small deviations for thederived parameters) was based on the findings of Shu andBevilacqua (19). They discovered that sequence preferences forstable triloops included aU-rich loop andC-G as the closing basepair. Based on these observations and the data reported here, thefollowing model was derived:

ΔG�37, triloop ¼ ΔG�37, i þΔG�37,UUU þΔG�37,C-G closure ð4ÞHere, ΔG�37,i is the triloop initiation term, 5.9 ( 0.1 kcal/mol,ΔG�37,UUU is a -1.2 ( 0.3 kcal/mol bonus for a UUU triloop,and ΔG�37,C-Gclosure is a -0.7 ( 0.2 kcal/mol bonus for triloopsadjacent to a C-G pair. Triloops adjacent to a G-C pair are notgiven the C-G bonus. Additional parameters and/or additionalbonuses may be discovered with additional experiments. Thismodel was used to predict the free energy contribution of thetriloops measured here and the triloops measured previously(16-18) (Table 2). On average, the predicted value was 0.3( 0.2kcal/mol different than the experimental value.

DISCUSSION

Database Searching.Due to the size and diversity of theRNAsecondary structure database that was searched, we have assumedthat the number and type of triloops found in this database arerepresentative of triloops found in naturally occurring RNA.

From the onset of this study, we anticipated that it would bedifficult to find triloop sequences that would outcompete bimo-lecular association of theRNA strand to forma duplexwith a 3�3 nucleotide internal loop (or smaller depending on the sequenceof the nucleotides) in 1 M NaCl. In fact, triloop formationoutcompeted bimolecular association with only two of the tenmost frequently occurring triloop sequences, and one of thosehad already been studied thermodynamically (16). Therefore, thetriloops studied here are not the most frequently occurringtriloops (Table 1, data set 1); however, all of the triloops studiedhere do occur in the database and do appear to outcompetebimolecular association of the RNA strand.

It is interesting to note that the most frequent triloop in thisdatabase was 50GGG30 (representing 13% of all triloops inthe database). In a similar database containing tetraloops,50GGGG30 was not found (39). In both the triloop andtetraloop database, a C-G base pair sat atop the list of closingbase pairs, representing 49% and 42% of all closing base pairsin tetraloops and triloops, respectively. When categorizing theloop nucleotides as purines and pyrimidines (Table 1, data set 4),it is interesting to note that loop sequences containing all purineswere the most common type for triloops (25%) as well as tetra-loops (35%).Thermodynamic Contributions of Triloops to Duplex

Thermodynamics. From the data in Table 2, it is evident thatthe stability of a triloop alone does not determine its frequency ofoccurrence. For example, the most stable triloop reported here(50CCUUG50, ΔG�37,triloop = 4.5 kcal/mol) is only the 45th mostcommon in the database. Interestingly, due to its abnormally lowdestabilizing free energy contribution, it is also the worstpredicted (using eq 4) of those reported here.

The sequence preferences observed here for stable triloopsare consistent with the general trends reported by Shu andBevilacqua (19). For example, triloops containing all uracils inthe loop were, on average, 0.9 kcal/mol more stable than othertriloop sequences. Triloops containing two uracils were, onaverage, 0.2 kcal/mol more stable than other triloop sequences;therefore, only triloops with three uracils are given a bonus in theproposed model (eq 4). Similarly, triloops adjacent to a C-G pairwere, on average, 0.6 kcal/mol more stable than triloops withother adjacent pairs. More specifically, when adjacent to a C-Gpair, both an 50AUA30 triloop and an 50AGA30 triloop are0.5 kcal/mol more stable than when adjacent to a G-C adjacentpair. Surprisingly, when adjacent to a C-G pair, a 50CUU30

triloop is 2.3 kcal/mol more stable than when adjacent to a G-Cpair. Although this difference in stability is almost four times theaverage stabilization by a C-G closing pair, this magnitude ofstabilization is not anomalous. When adjacent to a C-G pair, thetetraloop 50UUCG30 was found to be 2.3 kcal/mol more stablethan when adjacent to aG-C pair in 10mMNaCl (32). Similarly,when adjacent to a C-G pair, the 50UUA30 triloop found to be2.1 kcal/mol more stable than when adjacent to a G-C pair in10 mM NaCl (19).Updated Model for Predicting Thermodynamics of Tri-

loops. Because we have collected thermodynamic data for 24triloops that previously did not have experimental values,when predicting the free energy contributions of these triloopsin an RNA stem-loop, the experimental values can be used.For triloops that still do not have experimental values, thepredictive model (eq 4) can be utilized. It is unclear why50UUU30 is more stable than any other triloop sequence.Similarly, it is unclear why triloops with adjacent C-G pairsare more stable than triloops with adjacent G-C pairs. Struc-tural studies are currently underway to help to understandthese thermodynamic patterns. Additional studies with moretriloop sequences and adjacent base pairs may result in addi-tional free energy bonuses.

SUPPORTING INFORMATION AVAILABLE

A table listing all of the triloops found in the secondarystructure database and the type of RNA in which each wasfound. This material is available free of charge via the Internet athttp://pubs.acs.org.

9062 Biochemistry, Vol. 49, No. 42, 2010 Thulasi et al.

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