Optical Melting Studies Research Articles

Thermodynamic determination of RNA duplex stability in magnesium solutions

The prediction of RNA secondary structure and thermodynamics from sequence relies on free energy minimization and nearest neighbor parameters. Currently, algorithms used to make these predictions are based on parameters from optical melting studies performed in 1 M NaCl. However, many physiological and biochemical buffers containing RNA include much lower concentrations of monovalent cations and the presence of divalent cations. In order to improve these algorithms, thermodynamic data was previously collected for RNA duplexes in solutions containing 71, 121, 221, and 621 mM Na+. From this data, correction factors for free energy (ΔG°37) and melting temperature (Tm) were derived. Despite these newly derived correction factors for sodium, the stabilizing effects of magnesium have been ignored. Here, the same RNA duplexes were melted in solutions containing 0.5, 1.5, 3.0, and 10.0 mM Mg2+ in the absence of monovalent cations. Correction factors for Tm and ΔG°37 were derived to scale the current parameters to a range of magnesium concentrations. The Tm correction factor predicts the melting temperature within 1.2°C, and the ΔG°37 correction factor predicts the free energy within 0.30 kcalmol. These newly derived magnesium correction factors can be incorporated into algorithms that predict RNA secondary structure and stability from sequence.

Linking pH, Temperature, and K+ Concentration for DNA i-Motif Formation.

The conformation a particular DNA segment assumes depends upon its sequence context and the environment under which it is prepared. To complement our findings with G-rich sequences related to the human telomere, we have been investigating the pH induced transition from single strand to i-motif for sequences related to the human telomere C-rich strand. We have carried out titrations of (CCCTAA)4 from pH 7.0 to pH 5.0 at temperatures ranging from 15 to 45 °C at 115 mM K+ and at K+ concentrations ranging from 15 to 215 mM at 25 °C. Circular dichroism (CD) spectra were determined to monitor the transition. The pH at the midpoint of the proton induced transition, pHmp, is dependent upon both temperature and [K+]. Wyman-type plots of log K vs pH yielded linear correlations and the slopes of those lines, ΔQ, were also linearly dependent on [K+] and T. For these studies, ΔQ represents the minimum number of protons that must be added to the oligomer to induce the initial folding. These results are consistent with Le Chatelier's principle. Optical melting studies were also carried out for (CCCTAA)4 at pH 5.0 and [K+] ranging from 15 to 315 mM. Linear correlations between the temperature at the midpoint of the transition, Tm, and log [K+] allowed determination of the differential ion binding term, ΔnK+. These linkages between pH, temperature, and [K+] can be utilized to design i-motif forming DNA oligomers with highly tunable properties.

The loss of a hydrogen bond: Thermodynamic contributions of a non-standard nucleotide.

Non-standard nucleotides are ubiquitous in RNA. Thermodynamic studies with RNA duplexes containing non-standard nucleotides, whether incorporated naturally or chemically, can provide insight into the stability of Watson–Crick pairs and the role of specific functional groups in stabilizing a Watson–Crick pair. For example, an A-U, inosine•U and pseudouridine•A pair each form two hydrogen bonds. However, an RNA duplex containing a central I•U pair or central Ψ•A pair is 2.4 kcal/mol less stable or 1.7 kcal/mol more stable, respectively, than the corresponding duplex containing an A-U pair. In the non-standard nucleotide purine, hydrogen replaces the exocyclic amino group of A. This replacement results in a P•U pair containing only one hydrogen bond. Optical melting studies were performed with RNA duplexes containing P•U pairs adjacent to different nearest neighbors. The resulting thermodynamic parameters were compared to RNA duplexes containing A-U pairs in order to determine the contribution of the hydrogen bond involving the exocyclic amino group. Results indicate a loss of 1.78 kcal/mol, on average, when an internal P•U replaces A-U in an RNA duplex. This value is compared to the thermodynamics of a hydrogen bond determined by similar methods. Nearest neighbor parameters were derived for use in free energy and secondary structure prediction software.

Synthesis, Fluorescence Spectra, Redox Property and the DNA Binding Studies of 7-phenylacenaphtho[1,2-b]quinoxalin-7-ium chloride: Evidences of the Formation of Neutral Radical Analogue.

Reaction of acenaphthoquinone with N-phenyl-o-phenylenediamine in methanol in presence of HCl yielded 7-phenylacenaphtho[1,2-b]quinoxalin-7-ium chloride, [1][Cl]. [1][Cl] is brightly fluorescencent in dichloromethane (λex = 403 nm and λem = 442, 464, 488 nm) and water (λex = 408 nm and λem = 545 nm). Density functional theory (DFT) and time dependent (TD) DFT calculations on [1](+) at the B3LYP level of the theory elucidated that the origin of the lower energy excitation at around 400 nm is due to π → π(*) transition. [1](+) is redox active and exhibits a reversible cathodic wave at -0.66 V referenced to Fc(+)/Fc couple due to [1](+)/[1](•) redox couple. Electrogenerated neutral radical analogue [1](•) was characterized by electron paramagnetic resonance (EPR), UV-vis spectra and DFT calculations. DNA binding studies using the techniques of UV-vis absorption, fluorescence, circular dichroism (CD) spectra, viscosity, gel electrophoresis, hydrodynamic, isothermal titration calorimetry (ITC) and UV optical melting studies of [1][Cl] revealed that [1](+) is a strong DNA intercalator obeying neighbor exclusion principle. ITC experiment authenticated that the binding of [1](+) to DNA is entropy driven.

Phenazinium dyes safranine O and phenosafranine induce self-structure in single stranded polyadenylic acid: Structural and thermodynamic studies

The interaction of phenazinium dyes, safranine O and phenosafranine with single stranded polyadenylic acid was studied using spectroscopic viscometric and calorimetric techniques. Both dyes bind to polyadenylic acid strongly with association constant of the order of 105M−1. Safranine O showed higher affinity over phenosafranine. The binding induced conformational changes in polyadenylic acid, but the extent of change was much higher with safranine O. The bound safranine O molecules acquired strong induced circular dichroism spectra compared to the weak induced circular dichroism of phenosafranine. Fluorescence polarization, iodide quenching, viscosity results and energy transfer from bases to bound dyes suggested intercalation of the dye molecules to polyadenylic acid structure. The binding was entropy driven in both the cases. Circular dichroism and optical melting studies revealed cooperative melting profiles for dye–polyadenylic acid complexes that provided evidence for the formation of self-structured polyadenylic acid on dye binding. This structural reorganization was further confirmed by differential scanning calorimetry results.

Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction

BackgroundThe thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results.Methodology/Principal FindingsIsothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines.Conclusion/SignificanceFrom a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.

Isoquinoline Alkaloids and their Binding with DNA: Calorimetry and Thermal Analysis Applications

Alkaloids are a group of natural products with unmatched chemical diversity and biological relevance forming potential quality pools in drug screening. The molecular aspects of their interaction with many cellular macromolecules like DNA, RNA and proteins are being currently investigated in order to evolve the structure activity relationship. Isoquinolines constitute an important group of alkaloids. They have extensive utility in cancer therapy and a large volume of data is now emerging in the literature on their mode, mechanism and specificity of binding to DNA. Thermodynamic characterization of the binding of these alkaloids to DNA may offer key insights into the molecular aspects that drive complex formation and these data can provide valuable information about the balance of driving forces. Various thermal techniques have been conveniently used for this purpose and modern calorimetric instrumentation provides direct and quick estimation of thermodynamic parameters. Thermal melting studies and calorimetric techniques like isothermal titration calorimetry and differential scanning calorimetry have further advanced the field by providing authentic, reliable and sensitive data on various aspects of temperature dependent structural analysis of the interaction. In this review we present the application of various thermal techniques, viz. isothermal titration calorimetry, differential scanning calorimetry and optical melting studies in the characterization of drug-DNA interactions with particular emphasis on isoquinoline alkaloid-DNA interaction.

Thermodynamic Consequences of RNA Editing

RNA editing involves the deletion or insertion of nucleotides. In double stranded RNA, enzymes known as adenosine deaminases that act on RNA (ADARs) catalyze adenosine deamination, the conversion of adenine (A) to inosine (I). RNA editing enhances the complexity offered by the genome. ADAR concentrations are high in the nervous system and defects in editing can cause severe neurological disorders by targeting receptors such as those for serotonin and glutamate. Editing is expected to destabilize the double stranded RNA by creating regions of unpaired bases in the helices. Preference and selectivity of editing sites by ADARs is not fully understood but is attributed to the associated changes in thermodynamic stability of the RNA. In this study, we are measuring changes in RNA stability for edited and unedited RNA constructs based on the serotonin and glutamate receptor sites. The neighboring base pairs of adenine that is being edited influence the RNA stability, as expected based on the nearest neighbor models. Optical melting studies were performed on several different oligoribonucleotides constructs containing adenine or inosine to calculate the thermodynamic changes in RNA under different conditions. The nearest neighbor parameters are calculated using multiple linear regression models. This work is funded by NSF grant MCB‐0621509 (NG).

Effects for the Incorporation of Five-atom Thioacetamido Nucleic Acid (TANA) Backbone on Hybridization Thermodynamics and Kinetics of DNA Duplexes

Thermodynamic studies of nucleic acids serve not only to widen our understanding on the nature and strength of forces that stabilize nucleic acids in a myriad of structural states they assume but also to facilitate the development of databases that could be used to predict the stability and selectivity of probe/primer-sets required in a broad range of nucleic acid-based diagnostic and therapeutic protocols. In the current study, we investigated the effect of a novel, backbone-modified "thioacetamido linkage" (TANA) on thermodynamics of hybridization, binding kinetics, and conformation of a DNA duplex. The modification comprises of an extended five-atom amide (N3'-CO-CH2-S-CH2) linker, as opposed to four-atom phosphodiester linker backbone present in DNA. One to three TANA-substitutions have been introduced in the linker backbone of two thymidine residues of one of the strand of the DNA duplex. Using spectroscopic and calorimetric techniques, we observed that TANA destabilizes the DNA helix by lowering the favorable enthalpy parameter of duplex formation. TANA x DNA duplexes were found to adopt a conformation toward an A-type duplex as shown by circular dichroism spectroscopy studies. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, deltaCp, accompanying the duplex formation. The average deltaCp, change per duplex was found to be 832.5 cal mol(-1) K(-1), giving an average base-pair change of 59.5 cal (mol of base pairs)(-1) K(-1). Hybridization kinetic measurements using surface plasmon resonance indicated a decrease in binding affinity parameter (KA) that originates from higher dissociation rate constants (k(d)). Furthermore, optical melting studies showed that increasing the number of modifications results in a modest change in the number of counterions taken up during duplex formation.

Thermodynamics of DNA−RNA Heteroduplex Formation: Effects of Locked Nucleic Acid Nucleotides Incorporated into the DNA Strand

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue exhibiting enhanced hybridization efficiency toward complementary strand. The potential of LNA-based oligonucleotides has been sought to improve the selectivity and specificity of probe sets employed in detection and specific targeting of nucleic acids. We have evaluated the influence of "locked nucleic acid" residues on hybridization thermodynamics, counterions and hydration of DNA.RNA heteroduplex using spectroscopic and calorimetric techniques. One to three LNA substitutions have been introduced either at the adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) residues of the DNA strand. A complete thermodynamic profile for heteroduplex formation suggested that LNA-induced stabilization results from a favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, DeltaCp, accompanying the heteroduplex formation. Isothermal titration calorimetry measurements indicated an increase in binding affinity of the two strands as the LNA content of the heteroduplex is increased. Overall our result demonstrated that the effect of LNA-substitution at the thymine residue is more pronounced compared to the adenine residue. Furthermore, optical melting studies showed that, compared to an unmodified duplex, the formation of LNA-modified duplex is accompanied by a higher uptake of counterions and a lower uptake of water molecules. Our result, thus, presents a preliminary attempt toward the characterization of hybridization thermodynamics of the LNA-based probe-target sets, which will in turn aid in the selection of optimal conditions for hybridization experiments, and evaluation of the minimum probe-length required for hybridization and cloning experiments.

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4- N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4- N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.

Nearest Neighbor Parameters for Inosine·Uridine Pairs in RNA Duplexes

An enzyme family known as adenosine deaminases that act on RNA (ADARs) catalyzes adenosine deamination in RNA. ADARs act on RNA that is largely double-stranded and convert adenosine to inosine, resulting, in many cases, in an I x U pair. Thermodynamic parameters derived from optical melting studies are reported for a series of 14 oligoribonucleotides containing single I x U pairs adjacent to Watson-Crick pairs. In order to determine unique linearly independent nearest neighbor parameters for I x U pairs, four duplexes containing 3'-terminal I x U pairs and four duplexes containing 5'-terminal I x U pairs have also been thermodynamically characterized. This data was combined with previously published data of seven duplexes containing internal, terminal, or tandem I x U pairs from Strobel et al. [Strobel, S. A., Cech, T. R., Usman, N., and Beigelman, L. (1994) Biochemistry 33, 13824-13838] and Serra et al. [Serra, M. J., Smolter, P. E., and Westhof, E. (2004) Nucleic Acids Res. 32, 1824-1828]. On average, a duplex with an internal I x U pair is 2.3 kcal/mol less stable than the same duplex with an A-U pair, however, a duplex with a terminal I x U pair is 0.8 kcal/mol more stable than the same duplex with an A-U pair. Although isosteric with a G-U pair, on average, a duplex with an internal I x U pair is 1.9 kcal/mol less stable than the same duplex with a G-U pair, however, a duplex with a terminal I x U pair is 0.9 kcal/mol more stable than the same duplex with a G-U pair. Duplexes with tandem I x U pairs are on average 5.9 and 3.8 kcal/mol less stable than the same duplex with tandem A-U or tandem G-U pairs, respectively. Using the combined thermodynamic data and a complete linear least-squares fitting routine, nearest neighbor parameters for all nearest neighbor combinations of I x U pairs and an additional parameter for terminal I x U pairs have been derived.

Nearest neighbor parameters for Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides

Results from optical melting studies of Watson–Crick complementary heteroduplexes formed between 2′-O-methyl RNA and RNA oligonucleotides are used to determine nearest neighbor thermodynamic parameters for predicting the stabilities of such duplexes. The results are consistent with the physical model assumed by the individual nearest neighbor-hydrogen bonding model, which contains terms for helix initiation, base pair stacking and base pair composition. The sequence dependence is similar to that for Watson–Crick complementary RNA/RNA duplexes, which suggests that the sequence dependence may also be similar to that for other backbones that favor A-form RNA conformations.

Characterization of the Secondary Structure and Stability of an RNA Aptamer That Binds Vascular Endothelial Growth Factor

Thermal denaturation studies and spectroscopic studies were employed to investigate the secondary structure and stability of an RNA-PEG conjugate commercially called Macugen. The RNA aptamer is conjugated to a pegylated moiety, and the majority of its 2'-hydroxyl groups are methylated or otherwise modified. UV optical melting studies and differential scanning calorimetry (DSC) were carried out under different conditions to evaluate the effects of Na+ and oligomer concentrations on the stability of the secondary structure of the RNA oligomer. The results of these studies indicated that the T(m) of the RNA is independent of oligomer concentration but dependent on the salt concentration, in a predictable fashion. Further, the DSC melting profiles obtained under all conditions were highly reversible. Circular dichroism (CD) studies were determined under different salt concentrations, various RNA concentrations, and temperatures as well. Together, the thermal denaturation and CD studies provide evidence that the secondary structure of the RNA oligonucleotide is a stable hairpin at 25 degrees C and that the thermally induced hairpin to single strand transition is highly reversible.

The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA.

Influence of Sequence Context and Length on the Structure and Stability of Triplet Repeat DNA Oligomers

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.

Binding of cationic porphyrin to isolated and encapsidated viral DNA analyzed by comprehensive spectroscopic methods.

The complexation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with free and encapsidated DNA of T7 bacteriophage was investigated. To identify binding modes and relative concentrations of bound TMPyP forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. Spectral decomposition, fluorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of TMPyP, e.g., external binding and intercalation both in free and in encapsidated DNA. Optical melting studies revealed that TMPyP increases the strand separation temperature of both free and native phage DNA and does not change the phase transition temperature of phage capsid proteins. From these findings we concluded that TMPyP binding does not influence the protein structure and/or the protein-DNA interaction. A combined analysis of absorption spectra and fluorescence decay curves made possible the determination of concentrations of free, externally bound, and intercalated porphyrin. As a perspective, our results facilitate a qualitative analysis of the TMPyP binding process at various experimental conditions.

Thermodynamic parameters based on a nearest-neighbor model for DNA sequences with a single-bulge loop.

All 64 possible thermodynamic parameters for a single-bulge loop in the middle of a sequence were derived from optical melting studies. The relative stability of a single bulge depended on both the type of bulged base and its flanking base pairs. The contribution of the single bulge to helix stability ranged from 3.69 kcal/mol for a TAT bulge to -1.05 kcal/mol for an ACC bulge. Thermodynamics for 10 sequences with a GTG bulge were determined to test the applicability of the nearest-neighbor model to a single-bulge loop. Thermodynamic parameters for the GTG bulge and Watson-Crick base pairs predict, DeltaH degrees, DeltaS degrees, and T(M)(50 microM) values with average deviations of 3.0%, 4.3%, 4.7%, and 0.9 degrees C, respectively. The prediction accuracy was within the limits of what can be expected for a nearest-neighbor model. This certified that the thermodynamics for single-bulge loops can be estimated adequately using a nearest-neighbor model.

Photosensitized inactivation of T7 phage as surrogate of non-enveloped DNA viruses: efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.

A test of the model to predict unusually stable RNA hairpin loop stability.

To investigate the accuracy of a model [Giese et al., 1998, Biochemistry37:1094-1100 and Mathews et al., 1999, JMol Biol 288:911-940] that predicts the stability of RNA hairpin loops, optical melting studies were conducted on sets of hairpins previously determined to have unusually stable thermodynamic parameters. Included were the tetraloops GNRA and UNCG (where N is any nucleotide and R is a purine), hexaloops with UU first mismatches, and the hairpin loop of the iron responsive element, CAGUGC. The experimental values for the GNRA loops are in excellent agreement (deltaG degrees 37 within 0.2 kcal/mol and melting temperature (TM) within 4 degrees C) with the values predicted by the model. When the UNCG hairpin loops are treated as tetraloops, and a bonus of 0.8 kcal/mol included in the prediction to account for the extra stable first mismatch (UG), the measured and predicted values are also in good agreement (deltaG degrees 37 within 0.7 kcal/mol and TM within 3 degrees C). Six hairpins with unusually stable UU first mismatches also gave good agreement with the predictions (deltaG degrees 37 within 0.5 kcal/mol and TM within 8 degrees C), except for hairpins closed by wobble base pairs. For these hairpins, exclusion of the additional stabilization term for UU first mismatches improved the prediction (AG degrees 37 within 0.1 kcal/mol and TM within 3 degrees C). Hairpins with the iron-responsive element loop were not predicted well by the model, as measured deltaG degrees 37 values were at least 1 kcal/mol greater than predicted.