Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines.

A heptamer composed of C5-(1-propynyl) pyrimidines (Y(p)'s) is a potent and specific antisense agent against the mRNA of SV40 large T antigen (Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.; Froehler, B. C. Nat. Biotechnol. 1996, 14, 840-844). To characterize the role of the propynyl groups in molecular recognition, thermodynamic increments associated with substitutions in DNA:RNA duplexes, such as 5'-dCCUCCUU-3':3'-rGAGGAGGAAAU-5', have been measured by UV melting experiments. For nucleotides tested, an unpaired dangling end stabilizes unmodified and propynylated duplexes similarly, except that addition of a 5' unpaired rA is 1.4 kcal/mol more stabilizing on the propynylated, PODN:RNA, duplex than on the DNA:RNA duplex. Free energy increments for addition of single propynyl groups range from 0 to -4.0 kcal/mol, depending on the final number and locations of substitutions. A preliminary model for predicting the stabilities of Y(p)-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5'-dC(p)C(p)U(p)C(p)C(p)U(p)U(p)-3':3'-rGAGIAGGAAAU-5', destabilizes the duplex by 3.9 kcal/mol, compared to 1.7 kcal/mol for the same change within the unpropynylated duplex. This 2.2 kcal/mol difference is eliminated by removing a single propynyl group three base pairs away. CD spectra suggest that single propynyl deletions within the PODN:RNA duplex have position-dependent effects on helix geometry. The results suggest long-range cooperativity between propynyl groups and provide insights for rationally programming oligonucleotides with enhanced binding and specificity. This can be exploited in developing technologies that are dependent upon nucleic acid-based molecular recognition.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Unfolding thermodynamics of DNA pyrimidine triplexes with different molecularities

Nucleic acid oligonucleotides (ODNs), as drugs, present an exquisite selectivity and affinity that can be used in antigene and antisense strategies for the control of gene expression. In this work we try to answer the following question: How does the molecularity of a DNA triplex affect its overall stability and melting behavior? To this end, we used a combination of temperature-dependent UV spectroscopy and calorimetric (differential scanning calorimetry) techniques to investigate the melting behavior of DNA triplexes with a similar helical stem, TC+TC+TC+T/AGAGAGA/TCTCTCT, but formed with different strand molecularity. We determined standard thermodynamic profiles and the differential binding of protons and counterions accompanying their unfolding. The formation of a triplex is accompanied by a favorable free energy term, resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions, i.e., the folding of a particular triplex is enthalpy driven. The magnitude of the favorable enthalpy contributions corresponds to the number and strength of the base-triplet stacks formed, which are helped by stacking contributions due to the incorporation of dangling ends or loops. Triplex stability is in the following order: monomolecular > bimolecular > trimolecular; this is explained in terms of additional stacking contributions due to the inclusion of loops. As expected, acidic pH stabilized all triplexes by allowing protonation of the cytosines in the third strand; however, the percentage of protonation increases as the molecularity decreases. The results help to choose adequate solution conditions for the study of triplexes containing different ratios of CGC+ and TAT base triplets and to aid in the design of oligonucleotide sequences as targeting reagents that could effectively react with mRNA sequences involved in human diseases, thereby increasing the feasibility of using the antisense strategy for therapeutic purposes.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Comparative stability determination of oligonucleotide duplexes in gas and solution phase

The relative stability of RNA duplexes were determined in both solution and gas phase. Solution stability as determined by a spectrophotometric method indicated that the Watson-Crick duplexes were more stable than duplexes containing one GA mismatch or two tandem GA mismatches. Gas phase stability was determined using ESI-MS through variation of the collision energy in an ion trap. Stability curves similar to the melting curves obtained in solution were observed. The relative stability in gas phase differed, however, from that in solution. The duplexes with two tandem GA mismatches were found to be more stable than the Watson-Crick and single GA mismatch duplexes. The different trends observed in solution versus gas phase can be attributed to the primary means of interaction. In solution, stacking is expected to be the dominant interaction mode. In the gas phase, hydrogen bonding is expected to be the dominant interaction mode. Duplexes with tandem GA mismatches have the potential to undergo additional hydrogen bonding relative to the other duplexes which could account for their increased stability in the gas phase.     

Stability and structure of RNA duplexes containing isoguanosine and isocytidine.

Isoguanosine (iG) and isocytidine (iC) differ from guanosine (G) and cytidine (C), respectively, in that the amino and carbonyl groups are transposed. The thermodynamic properties of a set of iG, iC containing RNA duplexes have been measured by UV optical melting. It is found that iG-iC replacements usually stabilize duplexes, and the stabilization per iG-iC pair is sequence-dependent. The sequence dependence can be fit to a nearest-neighbor model in which the stabilities of iG--iC pairs depend on the adjacent iG--iC or G--C pairs. For 5'-CG-3'/3'-GC-5' and 5'-GG-3'/3'-CC-5' nearest neighbors, the free energy differences upon iG-iC replacement are smaller than 0.2 kcal/mol at 37 degrees C, regardless of the number of replacements. For 5'-GC-3'/3'-CG-5', however, each iG--iC replacement adds 0.6 kcal/mol stabilizing free energy at 37 degrees C. Stacking propensities of iG and iC as unpaired nucleotides at the end of a duplex are similar to those of G and C. An NMR structure is reported for r(CiGCGiCG)(2) and found to belong to the A-form family. The structure has substantial deviations from standard A-form but is similar to published NMR and/or crystal structures for r(CGCGCG)(2) and 2'-O-methyl (CGCGCG)(2). These results provide benchmarks for theoretical calculations aimed at understanding the fundamental physical basis for the thermodynamic stabilities of nucleic acid duplexes.     

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.     

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.     

Theoretical analysis of antisense duplexes: determinants of the RNase H susceptibility

The structure and dynamic properties of different antisense related duplexes (DNA x RNA, 2'O-Me-DNA x RNA, 2'F-ANA x RNA, C5(Y)-propynyl-DNA x RNA, ANA x RNA, and control duplexes DNA x DNA and RNA x RNA) have been determined by means of long molecular dynamics simulations (covering more than 0.5 micros of fully solvated unrestrained MD simulation). The massive analysis presented here allows us to determine the subtle differences between the different duplexes, which in all cases pertain to the same structural family. This analysis provides information on the molecular determinants that allow RNase H to recognize and degrade some of these duplexes, whereas others with apparently similar conformations are not affected. Subtle structural and deformability features define the key properties used by RNase H to discriminate between duplexes.     

Understanding the recognition mechanism of protein-RNA complexes using energy based approach

Protein-RNA interactions perform diverse functions within the cell. Understanding the recognition mechanism of protein-RNA complexes is a challenging task in molecular and computational biology. In this work, we have developed an energy based approach for identifying the binding sites and important residues for binding in protein-RNA complexes. The new approach considers the repulsive interactions as well as the effect of distance between the atoms in protein and RNA in terms of interaction energy, which are not considered in traditional distance based methods to identify the binding sites. We found that the positively charged, polar and aromatic residues are important for binding. These residues influence to form electrostatic, hydrogen bonding and stacking interactions. Our observation has been verified with the experimental binding specificity of protein-RNA complexes and found good agreement with experiments. Further, the propensities of residues/nucleotides in the binding sites of proteins/RNA and their atomic contributions have been derived. Based on these results we have proposed a novel mechanism for the recognition of protein-RNA complexes: the charged and polar residues in proteins initiate recognition with RNA by making electrostatic and hydrogen bonding interactions between them; the aromatic side chains tend to form aromatic-aromatic interactions and the hydrophobic residues aid to stabilize the complex.     

2-aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligonucleotides

2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.     

Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.     

Spermine moiety attached to the C-5 position of deoxyuridine enhances the duplex stability of the phosphorothioate DNA/complementary DNA and shows the susceptibility of the substrate to RNase H

Novel phosphorothioate-modified oligodeoxynucleotides (S-ODNs) containing a deoxyuridine derivative bearing a spermine moiety at the C-5 position were synthesized. The study of the thermal stability and the thermodynamic stability showed that the modified S-ODNs have been able to form the stable duplexes with the complementary DNA. It was also found that the duplex composed of the modified S-ODN and its complementary RNA strand is the substrate for Escherichia coli RNase H, and the cleavage of the RNA strand by the enzyme was almost similar as in the case of the unmodified one.     

Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands

To understand the influences of the ribonucleotide on a duplex conformation and its stability, we systematically studied the CD spectra and the thermodynamics of nucleic acid duplexes formed by the chimeric RNA-DNA strand in which ribonucleotides and deoxyribonucleotides were covalently attached. It was found that the duplex stability was context-dependent and independent of the number of ribonucleotides in the chimeric strand, whereas the CD spectra showed less overall structural perturbation by the chimeric junctions. Combining the results of the CD and the thermodynamic data revealed a stability-structure relationship for the duplexes. Importantly, DeltaG(o)37 values estimated for the chimeric junction formation in the RNA-DNA/DNA and the RNA-DNA/RNA duplexes were close to those of RNA/DNA and RNA/RNA interactions, respectively. Furthermore, DeltaG(o)37s of the DNA-RNA/DNA and DNA-RNA/DNA-RNA junctions were similar to those of the DNA duplex, and the values of DNA-RNA/RNA-DNA were similar to those of the DNA/RNA. The thermodynamic analyses suggest that the 5'-nucleotide may be the crucial factor that determines the stability at the chimeric junction. Our results not only suggest influences of the ribonucleotide on a duplex conformation and its stability but also are useful for the design of RNA-DNA chimeric strands applicable to biotechnology.     

Free energy simulations of uncatalyzed DNA replication fidelity: structure and stability of T.G and dTTP.G terminal DNA mismatches flanked by a single dangling nucleotide

A reference system for DNA replication fidelity was studied by free energy perturbation (FEP) and linear interaction energy (LIE) methods. The studied system included a hydrated duplex DNA with the 5'-CG dangling end of the templating strand, and dCTP4-.Mg2+ or dTTP4-.Mg2+ inserted opposite the dangling G to form a correct (i.e., Watson-Crick) or incorrect (i.e., wobble) base pair, respectively. The average distance between the 3'-terminal oxygen of the primer strand and the alpha-phosphorus of dNTP was found to be 0.2 A shorter for the correct base pair than for the incorrect base pair. Binding of the incorrect dNTP was found to be disfavored by 0.4 kcal/mol relative to the correct dNTP. We estimated that improved binding and more near-attack configurations sampled by the correct base pair should translate in aqueous solution and in the absence of DNA polymerase into a six times faster rate for the incorporation of the correct dNTP into DNA. The accuracy of the calculated binding free energy difference was verified by examining the relative free energy for melting duplex DNA containing GC and GT terminal base pairs flanked by a 5' dangling C. The calculated LIE and FEP free energies of 1.7 and 1.1 kcal/mol, respectively, compared favorably with the experimental estimate of 1.4 kcal/mol obtained using the nearest neighbor parameters. To decompose the calculated free energies into additive electrostatic and van der Waals contributions and to provide a set of rigorous theoretical data for the parametrization of the LIE method, we suggested a variant of the FEP approach, for which we coined a binding-relevant free energy (BRFE) acronym. BRFE approach is characterized by its unique perturbation pathway and by its exclusion of the intramolecular energy of a rigid part of the ligand from the total potential energy.     

Synthesis of 2',4'-propylene-bridged (carba-ENA) thymidine and its analogues: the engineering of electrostatic and steric effects at the bottom of the minor groove for nuclease and thermodynamic stabilities and elicitation of RNase H

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH(2)/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C═N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON:RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance but 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH(2) substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase H1 with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.     

A distance ruler for RNA using EPR and site-directed spin labeling

As a basic model study for measuring distances in RNA molecules using continuous wave (CW) EPR spectroscopy, site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined. The spin labels were attached to the 2'-NH2 positions of appropriately placed uridines in the duplexes, and interspin distances were measured from both molecular dynamics simulations (MD) and Fourier deconvolution methods (FD). The 10-mer duplexes have interspin distances ranging from 10 A to 30 A based on MD; however, dipolar line broadening of the CW EPR spectrum is only observed for the RNAs for predicted interspin distances of 10-21 A and not for distances over 25 A. The conformational changes in TAR (transactivating responsive region) RNA in the presence and in the absence of different divalent metal ions were monitored by measuring distances between two nucleotides in the bulge region. The predicted interspin distances obtained from the FD method and those from MD calculations match well for both the model RNA duplexes and the structural changes predicted for TAR RNA. These results demonstrate that distance measurement using EPR spectroscopy is a potentially powerful method to help predict the structures of RNA molecules.     

C-F...H-C hydrogen bonds in ribonucleic acids

We report the synthesis of 1'-deoxy-1'-(benzimidazol-1-yl)-beta-D-ribofuranose 7 and 1'-deoxy-1'-phenyl-beta-D-ribofuranose 2. With these two ribonucleoside analogues we have a set of nine different RNA building blocks in hand, which are isostere to the natural bases. Now it is possible to investigate their duplex stabilizing forces. These forces are hydrogen bonds, base stacking, and solvation. The phosphoramidites of all building blocks were incorporated into a 12mer RNA, and the resulting RNA duplexes were investigated by UV- and CD-spectroscopy. We found that some of the RNA analogues are universal bases. The best universal bases with the lowest destabilization and the smallest discrimination between the natural bases are 1 (B) and 9 (E). On the basis of UV measurements we determined the melting points and the thermodynamic data. We were able to show that there are no hydrogen bonds between the natural bases and the RNA analogues. From thermodynamic data we calculated the contributions for base stacking and solvation of all modified building blocks. Comparison of calculated and measured data of double modified base pairs in 12mer RNA duplexes showed a further duplex stabilizing force in base pairs containing fluorine atoms at the Watson-Crick binding site. This stabilizing force can be defined as C-F.H-C hydrogen bond as is observed in crystal structures of 1'-deoxy-1'-(4-fluorophenyl)-beta-D-ribofuranose.     

On the stability of double stranded nucleic acids

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.     

Measurement of the effect of monovalent cations on RNA hairpin stability

Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A.U and G.C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (<15 bp) duplexes and hairpins in 1 M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks fluctuation theorem. We observed the following: (1) In most cases, the nearest-neighbor model accurately predicted the free energy of folding at 1 M NaCl. (2) Free energy was proportional to the logarithm of salt concentration. (3) Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. (4) Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G.C helices were unaffected by changing salt concentration, A.U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the U.C.U bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.