Thermodynamic stability of the 5' dangling-ended DNA hairpins formed from sequences 5'-(XY)2GGATAC(T)4GTATCC-3', where X, Y = A, T, G, C

Expressions for the partition function Q (T) of DNA hairpins are presented. Calculations of Q (T), in conjunction with our previously reported numerically exact algorithm [T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight (1990) Biopolymers, 29, 1715-1734], yield a numerical method to evaluate the temperature dependence of the transition enthalpy, entropy, and free energy of a DNA hairpin directly from its optical melting curve. No prior assumptions that the short hairpins melt in a two-state manner are required. This method is then applied in a systematic manner to investigate the stability of the six basepair duplex stem 5'-GGATAC-3' having four-base dangling single-strand ends with the sequences (XY)2, where X, Y = A, T, G, C, on the 5' end and a T4 loop on the 3' end. Results show that all dangling ends of the sample set stabilize the hairpin against melting. Increases in transition temperatures as great as 4.0 degrees C above the blunt-ended control hairpin were observed. The hierarchy of the hairpin transition temperatures is dictated by the identity of the first base of the dangling end adjoining the duplex in the order: purine greater than T greater than C. Calculated melting curves of every hairpin were fit to experimental curves by adjustment of a single parameter in the numerically exact theoretical algorithm. Exact fits were obtained in all cases. Experimental melting curves were also calculated assuming a two-state melting process. Equally accurate fits of all dangling-ended hairpin melting curves were obtained with the two-state model calculation. This was not the case for the melting curve of the blunt-ended hairpin, indicating the presence of a four-base dangling-end drives hairpin melting to a two-state process. Q (T) was calculated as a function of temperature for each hairpin using the theoretical parameters that provided calculated curves in exact agreement with the experimentally obtained optical melting curves. From Q (T), the temperature dependence of the transition enthalpy delta H, entropy delta S, and free energy delta G were calculated for every hairpin providing a quantitative assessment of the effects of dangling ends on hairpin thermodynamics. Comparisons of our results are made with those of the Breslauer group [M. Senior, R. A. Jones, and K. J. Breslauer (1988) Biochemistry 27, 3879-3885] on the T2 5' dangling-ended d(GC)3 duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of melting transitions of the DNA hairpins formed from the oligomer sequences d[GGATAC(X)4GTATCC] (X = A, T, G, C)

Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3 degrees C for the T4 loop hairpin to 55.3 degrees C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop formation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions. Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop.(ABSTRACT TRUNCATED AT 400 WORDS)     

Melting studies of short DNA hairpins: Influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self‐complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature‐induced melting transitions in buffered 115 mM Na⁺ solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5°C and are concentration independent over strand concentrations of 0.5 to 260 μM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, ΔH_cal, ΔS_cal, and ΔG_cal, were determined from DSC measurements. Model dependent transition parameters, ΔH_vH, ΔS_vH, and ΔG_vH were estimated from a van't Hoff (two‐state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, ΔH_cal (ΔH_vH) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy ΔS_cal (ΔS_vH) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies ΔG_cal (ΔG_vH) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two‐state model reveal that 10 of the 28 hairpins display non‐two‐state melting behavior. The database of sequence‐dependent melting free energies obtained for the hairpins was employed to extract a set of n‐n (nearest‐neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest‐neighbor loop sequence parameters. © 1999 John Wiley & Sons, Inc. Biopoly 50: 425–442, 1999     

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.     

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases.     

Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed -1 ribosomal frameshifting.

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseudoknot from the gag-pro junction of mouse intracisternal A-type particles (mIAP), an endogenous retrovirus, has been determined through analysis of dual optical wavelength, equilibrium thermal melting profiles and differential scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot proposed to have structural features in common with the gag-pro frameshifting pseudoknots from simian retrovirus-1 (SRV-1) and mouse mammary tumor virus (MMTV). In particular, the mIAP pseudoknot is proposed to contain an unpaired adenosine base at the junction of the two helical stems (A15), as well as one in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins and compensatory base-pair substitutions incorporated into helical stem 2 was used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in two closely coupled low-enthalpy transitions at low tmin which the stem 3' to A35, unfolds first, followed by unfolding of the remainder of the helical stem. The third unfolding transition is associated with some type of stacking interactions in the stem 1 hairpin loop not present in the pseudoknot. The fourth transition is assigned to unfolding of stem 1. In all RNAs investigated, DeltaHvH approximately DeltaHcal, suggesting that DeltaCpfor unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the stem 2 unfolding transition in the context of both the wild-type and DeltaA35 mutant RNAs only slightly, by DeltaDeltaG degrees approximately 1 kcal mol-1(at 37 degrees C). The DeltaA15 RNA is considerably more susceptible to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabilization of the molecule. Interestingly, substitution of the nine loop 2 nucleotides with uridine residues induces a more pronounced destabilization of the molecule (DeltaDeltaG degrees approximately 2.0 kcal mol-1), a long-range, non-nearest neighbor effect. These findings provide the thermodynamic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability.     

Increase in hybridization rates with oligodeoxyribonucleotides containing locked nucleic acids

Locked nucleic acids (LNAs) incorporated into either stable single stranded oligonucleotides containing tetraloops or their complements have been found to increase second order hybridization rate constants by an order of magnitude compared to the all-DNA hybridization rate constants. Model sequences composed of 20 bases in length that can form hairpins due to a stable GAAA tetraloop were used where LNAs were substituted for the nucleotides in the loop, stem, or end regions of the strand and in the complementary strand. Substitution of the LNAs to the loop predictably raised the melting temperatures of the duplex however, the hybridization rates between the tetraloop and the complementary sequence also increased. In contrast, when LNAs were substituted in the stem, the hybridization rate decreased implying the formation of a more stable hairpin. Substitution of LNAs into the end region of the sequence had little effect on the hybridization rate constants although melting temperatures still showed a predictable increase. Rates also increased when LNAs were substituted into complementary strands of DNA tetraloops. The increase in hybridization rate constant is being attributed to changes in the structure of the stable single strands.     

Oligonucleotides containing substituted 4-nitroindoles: Synthesis and study of their DNA duplexes

The synthesis of oligonucleotides containing 1-(2-deoxy-β-D-ribofuranosyl)-2-methyl-4-nitroindole and 1-(2-deoxy-β-D-ribofuranosyl)-2-phenyl-4-nitroindole is described. The synthesized modified oligonucleotides were used for studying the stability of intermolecular DNA duplexes with one unnatural strand and for evaluation of discriminating potential of 2-methyl-and 2-phenyl-4-nitroindoles toward nucleic bases. For comparison, an unmodified oligonucleotide and oligonucleotides bearing 5-nitroindole were used. It was shown that 2-methyl-4-nitroindole was only insignificantly inferior in stability to 5-nitroindole and characterized by a similar discriminating potential. 2-Phenyl-4-nitroindole provided a more pronounced duplex destabilization, the discrimination toward natural bases being decreased.     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.     

Oligodeoxynucleotides containing substituted 4-nitroindoles: synthesis and study of their DNA duplexes

The synthesis of oligonucleotides containing 1-(2-deoxy-beta-D-ribofuranosyl)-2-methyl-4-nitroindole and 1-(2-deoxy-beta-D-ribofuranosyl)-2-phenyl-4-nitroindole is described. The synthesized modified oligonucleotides were used for studying the stability of intermolecular DNA duplexes with one unnatural strand and for evaluation of discriminating potential of 2-methyl- and 2-phenyl-4-nitroindoles toward nucleic bases. For comparison, an unmodified oligonucleotide and oligonucleotides bearing 5-nitroindole were used. It was shown that 2-methyl-4-nitroindole was only insignificantly inferior in stability to 5-nitroindole and characterized by a similar discriminating potential. 2-Phenyl-4-nitroindole provided a more pronounced duplex destabilization, the discrimination toward natural bases being decreased. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.     

Structural similarities and differences between H1- and H2-family DNA minihairpin loops: NMR studies of octameric minihairpins

TheDNA sequences 5′-d(CGC-AC-GCG)-3′ (HPAC), 5′-d(CGC-AA-GCG)-3′ (HPAA), 5′-d(CGC-TC-GCG)-3′ (HPTC), and 5′-d(CGC-CT-GCG)-3′ (HPCT), were studied by means of nmr spectroscopy. At low DNA concentration and no added salt all four molecules adopt a minihairpin structure, containing three Watson–Crick base pairs and a two-residue loop. The structure of the HPAC hairpin is based on quantitative distance restraints, derived by a full relaxation matrix approach (iterative relaxation matrix approach), together with torsion angles obtained from coupling constant analysis. The loop folding is of the H1-family type, characterized by continuous 3′-5′ stacking of the loop bases on the duplex stem. The structure of the HPAA hairpin is similar to that of HPAC, but is more flexible and has a lower thermodynamic stability (T_m 326 K vs 320 K). According to “weakly” distance-constrained simulations in water on the HPAC minihairpin, the typical H1-family loop folding remains intact during the simulation. However, residue-based R factors of simulated nuclear Overhauser effect spectroscopy spectra, free molecular dynamics simulations in vacuo, and unusual chemical shift profiles indicate partial destacking of the loop bases at temperatures below the overall melting midpoint. The dynamic nature of the loop bases gives insight into the geometrical tolerances of stacking between bases in H1-family minihairpin loops. The HPTC and HPCT minihairpins, both containing a pyrimidine base at the first position in the loop, adopt a H2-family type folding, in which the first loop base is loosely bound in the minor groove and the second loop base is stacked upon the helix stem. The thermal stability for these two hairpins corresponds to 327–329 K, but depends on local base sequence. Preference for the type of folding depends on a single substitution from a pyrimidine (H2 family) to a purine (H1 family) at the first position of the miniloop and is explained by differences in base stacking energies, steric size, and the number of possible candidates for hydrogen bonds in the minor groove. In view of newly collected data, previous models of the H1-family and H2-family hairpins had to be revised and are now compatible with the reported HPTC and HPAC structures. The structural difference between the refined structure of HPAC and HPTC show that a conversion between H1-family and H2-family hairpins is geometrically possible by a simple pivot point rotation of 270° along two torsion angles, thereby swiveling the first loop base from a stacked position in a H1-family folding toward a position in the minor groove in a H2-family folding. The second loop residue subsequently shifts to the position of the first base in a concerted fashion. © 1998 John Wiley & Sons, Inc. Biopoly 46: 375–393, 1998     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.     

Studies of DNA dumbbells. VI. Analysis of optical melting curves of dumbbells with a sixteen-base pair duplex stem and end-loops of variable size and sequence

Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5′-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3′ linked on both ends by single-strand loops of A_t or C_t sequences (˛ = 2, 3, 4, 6, 8, 10, 14), T_t sequences (˛ = 2, 3, 4, 6, 8, 10), and G_t sequences (t = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM Na⁺. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation ΔG_loop(n > t) and single-strand circles, ΔG_cir(N) as a function of end-loop size, t = 2-14, circle size, N = 32 + 2_t, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy ΔH, entropy ΔS, and free energy ΔG, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na⁺]. Since the multistate analysis is based on the numerically exact model, and considers a statistically significant number of theoretically possible partially melted states, it does not require prior assumptions regarding the nature of the melting transition, i.e., whether or not it occurs in a two-state manner. For comparison with the multistate analysis, thermodynamic transition parameters were also evaluated directly from experimental melting curves assuming a two-state transition and using the graphical van't Hoff analysis. Comparisons between results of the multistate and two-state analyses suggested dumbbells with loops comprised of six or fewer residues melted in a two-state manner, while the melting processes for dumbbells with larger end-loops deviate from two-state behavior.Dependence of thermodynamic parameters on[Na⁺] as a function of loop size suggests single-strand end-loops have different counterion binding properties than the melted circle. Results are compared with those obtained in an earlier study of dumbbells with the slightly different stem sequence 5'-G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C-3' linked on the ends by T loops (˛ = 2,3,4,6,8,10,14).© 1996 John Wiley &Sons, Inc.     

Characterization of the structure and melting of DNAs containing backbone nicks and gaps

A DNA molecule containing a gap (a missing phosphate) has been examined and compared to two other molecules of the same sequence, one containing a nick (a phosphorylated gap) and the other a normal duplex containing no break in the backbone. A second gapped sequence was also compared to a normal duplex of the same sequence. The molecules containing nicks or gaps were generated as dumbbell molecules, short helices closed by a loop at each end. The dumbbells were formed by the association of two hairpins with self-complementary dangling 5'-ends. Nuclear magnetic resonance was used to monitor the melting transition and to probe structural differences between molecules. Under the conditions used here no change in stability was observed upon phosphorylation of the gap. Structural changes upon phosphorylation of a gap or closure of a nick were minimal and were localized to the region immediately around the gap or nick. Two transitions can be observed as a gapped or nicked molecule melts, although the resolution of the two transitions varies with the salt concentration. At moderate to high salt (greater than or equal to 30 mM) the molecule melts essentially all at once. At low salt the two transitions occur at temperatures that differ by as much as 15 degrees C. In addition, comparison with other NMR melting studies indicates that the duplex formed by the overlap of the dangling ends of the hairpins is stabilized relative to a free duplex of the same sequence, probably by stacking onto the hairpin stem.     

Extra stable 2',5'-linked RNA loops

We prepared hairpins that differ in the connectivity of phosphodiester linkages in the loop (RNA vs 2', 5'-RNA). We find that the stability of the extra stable RNA hairpin 5'-rGGAC(UUCG)GUCC-3' is the same as that observed for the hairpin containing a 2',5'RNA loop, i.e. 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U2'p5'U2'p5' C2'p5'G2'p5'). Also significant is the finding that when the stem is duplex DNA, duplex 2',5'-RNA, or DNA:2',5'-RNA, hairpins with the UUCG loop are more stable than those with UUCG loop.     

5-Nitroindole as an universal base analogue.

4-, 5- and 6-Nitroindole have been investigated and compared with 3-nitropyrrole as universal bases in oligodeoxynucleotides. Of these the 5-nitroindole derivative was found to be superior giving higher duplex stability, and behaving indiscriminately towards each of the four natural bases in duplex melting experiments. 3-Nitropyrrole, whilst not discriminating between the natural bases, was found to lead to considerable destabilisation of the duplexes, particularly when multiple substitutions were made, in contrast to the 5-nitroindole nucleoside.     

一种用于优化PCR引物设计的短链DNA熔点分析方法

目前,PCR引物设计主要依赖于软件对引物熔点的模拟计算,而PCR退火条件的优化需进行不同条件下的扩增实验。为开发一种可高效、精确评价引物和确定退火条件的方法,本研究采用高分辨率熔解曲线（high resolution melting,HRM）测定技术直接分析短链DNA的熔点,用于引物优劣性的评价,并为退火条件的优化提供参考。本文用HRM法直接测定了非完全互补的双链DNA以及DNA发卡结构的熔点,结果显示：（1）与完全互补的双链DNA相比,较为稳定的单碱基错配A?G、G?G和T?G的熔点只降低2℃ ~ 3℃,部分双碱基错配的熔点只降低4℃ ~ 6℃,单碱基突出熔点只降低4℃~ 5℃。因此,如果采用的退火温度不当,部分错配的非目的模板可能会被扩增。（2）即使发卡结构的茎干区只有6 bp,当其环区碱基少于10 nt时,其熔点也可达到60℃以上。此外,环区的长度对发卡熔点也有较大影响。根据本研究结果发现,引物设计时应尽量避免模板引物结合区同其邻近的30 nt碱基有6 bp以上的互补部分。综上所述,本研究证明HRM熔点法是一种高效评价引物及确定退火温度的方法。     

Transition characteristics and thermodynamic analysis of DNA duplex formation: a quantitative consideration for the extent of duplex association

Transition characteristics and thermodynamic properties of the single-stranded self-transition and the double-stranded association were investigated and analyzed for 9-, 15- and 21-bp non-self-complementary DNA sequences. The multiple transition processes for the single-stranded self-transition and the double-stranded association were further put forth. The experimental results confirmed that the double-stranded association transition was generally imperfect and the thermodynamic properties of the single-stranded self-transition would exert an influence on a duplex formation. Combining ultraviolet melting experiments in various molar ratios, the extent of duplex association was estimated for three double-stranded DNAs. In our experimental range, the extent of duplex association decreases with increasing the number of base pairs in DNA sequences, which suggest that the short oligonucleotides may proceed in a two-state transition while the long oligonucleotides may not. When the extent of duplex association was considered, the true transition enthalpies of a duplex formation derived from UV and differential scanning calorimetry measurements were in good agreement.     

Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation delta Gloop (n) and single-strand circles delta Gcircle (N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy delta H degrees and entropy delta S degrees for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, pp. 377-399).(ABSTRACT TRUNCATED AT 400 WORDS)