Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability

Kierzek, Ryszard; Caruthers, Marvin H.; Longfellow, Carl E.; Swinton, David; Turner, Douglas H.; Freier, Susan M.

doi:10.1021/bi00372a009

Cited by 148 publications

(106 citation statements)

References 26 publications

(36 reference statements)

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“…For example, for the chemically synthesized hairpin having an AA mismatch next to the C G closing base pair, AG 0 (stem) = AG 0 ( 9) + AG3 ( ) = -2.9-3.4 = -6.3 kcal/mol, and the predicted AG°(hairpin) = AG' (loop of 6) + AG' (stem) + AGO A DISCUSSION Current models for predicting the secondary structural stability of RNA employ many simplifying assumptions, particularly for loop regions (1,5). Improved methods for the synthesis of RNA molecules of defined sequence allow testing the validity of these assumptions (9)(10)(11)(12)(13) Comparison of loops with first mismatches of AA and GA (Table I) suggests this model is also oversimplified. Studies of RNA and DNA hairpin loops with 4 nucleotides also reveal a significant sequence dependence (22,(24)(25)(26).…”

Section: Resultsmentioning

confidence: 99%

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RNA hairpin loop stability depends on closing base pair

Serra¹,

Lyttle²,

Axenson³

et al. 1993

Nucl Acids Res

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Section: Resultsmentioning

confidence: 99%

“…This was based on a comparison of hairpins formed by A4GCnCU4 (7) and AnU6 (8) (9)(10)(11). GGUAUAAUAGCC was synthesized with the 2 hydroxyl protected as the tetrahydropyranyl ether (12). pppGGCAUAAUAGCC and pppGGCGUAAUA-GCC were synthesized with the T7 RNA polymerase method of Milligan et al (13).…”

Section: Introductionmentioning

confidence: 99%

RNA hairpin loop stability depends on closing base pair

Serra¹,

Lyttle²,

Axenson³

et al. 1993

Nucl Acids Res

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“…Free energy increments for hydrogen-bonded AU, GC, and GU pairs are based on the nearest-neighbor model (5,7,8,18). Measured free energy increments for terminal hydrogen-bonded GU pairs are an average of 0.2 kcal/mol more favorable than internal GU mismatches (19).…”

Section: Methodsmentioning

confidence: 99%

“…(i) New methods for synthesizing RNA make it possible to obtain model systems with a large variety of sequence (5,6). This technique has led to measurements of improved parameters and the realization that non-base-paired nucleotides contribute sequence-dependent interactions that stabilize secondary structure (7,8).…”

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confidence: 99%

Improved predictions of secondary structures for RNA.

Jaeger

Turner

Zuker

1989

Proc. Natl. Acad. Sci. U.S.A.

774

541

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The accuracy of computer predictions of RNA secondary structure from sequence data and free energy parameters has been increased to roughly 70%. Performance is judged by comparison with structures known from phylogenetic analysis. The algorithm also generates suboptimal structures. On average, the best structure within 10% of the lowest free energy contains roughly 90% of phylogenetically known helixes. The algorithm does not include tertiary interactions or pseudoknots and employs a crude model for singlestranded regions. The only favorable interactions are base pairing and stacking of terminal unpaired nucleotides at the ends of helixes. The excellent performance is consistent with these interactions being the primary interactions determining RNA secondary structure.RNA is important for functions such as catalysis, RNA splicing, regulation of transcription and translation, and transport of proteins across membranes (1). Many RNA sequences are known. Determination of secondary structures, however, is difficult. Thermodynamics has been applied to predict RNA secondary structure from sequence (2, 3), but with modest success. Predictions of suboptimal structures make the method more useful (4). We report combining several recent advances to improve predictions of RNA secondary structures. Three advances are incorporated in this work. (i) New methods for synthesizing RNA make it possible to obtain model systems with a large variety of sequence (5, 6). This technique has led to measurements of improved parameters and the realization that non-base-paired nucleotides contribute sequence-dependent interactions that stabilize secondary structure (7, 8). (ii) A computer algorithm has been developed that allows incorporation of non-base-paired interactions in the prediction of optimal and suboptimal secondary structures (9). (iii) Several RNA secondary structures have been determined by phylogeny (10-15). Comparison of predicted and known structures allows optimization of parameters that have not been measured. Resultant predictions appear sufficiently reliable to aid planning and interpretation of experiments on RNA. MATERIALS AND METHODSThermodynamic Parameters. When possible, free energy increments at 370C, AG037, were taken from experiments in 1 M NaCl. For fully base-paired regions, experiments on dGCATGC indicate that 1 M NaCl mimics solutions containing 1-100 mM Mg2+ in the presence of 0.15-1 M NaCl (16).Relatively few experiments are available for loop structures, and, therefore, little is known about interactions determining loop stability. This situation forced approximations. (i) Jacobson-Stockmayer theory (17) was used to extrapolate the length dependence of AG37 for bulge, hairpin, and internal loops: AG0(n) = AG(nmax) + 1.75 RTln (n/nmax).For this equation n is the number of unpaired nucleotides in the loop, nma is the maximum-length loop for which experimental data is available, R is the gas constant (1.987 cal mol'lK-l; 1 cal = 4.184 J), and T is the temperature in K (310.15 K for 370C). (ii) When...

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“…30 Synthesized 7 decamers were desalted with a C-18 Sep-pak cartridge column (Waters). The final purity of the decamers was confirmed to be greater than at least 98% by HPLC.…”

Section: Methodsmentioning

confidence: 99%