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Nucleic Acids Research, 2002, Vol. 30, No. 18 4088-4093
© 2002 Oxford University Press

Melting studies of dangling-ended DNA hairpins: effects of end length, loop sequence and biotinylation of loop bases

Peter V. Riccelli, Kathleen E. Mandell and Albert S. Benight*

Department of Chemistry, M/C 111, Science and Engineering Building, University of Illinois at Chicago, 845 West Taylor Street, Room 4500, Chicago, IL 60607-7061, USA

*To whom correspondence should be addressed. Tel: +1 312 996 0774; Fax: +1 312 996 0431; Email: abenight{at}uic.edu

Received March 2, 2002; Revised July 11, 2002; Accepted July 20, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The effects of 3' single-strand dangling-ends of different lengths, sequence identity of hairpin loop, and hairpin loop biotinylation at different loop residues on DNA hairpin thermodynamic stability were investigated. Hairpins contained 16 bp stem regions and five base loops formed from the sequence, 5'-TAGTCGACGTGGTCC-N5-GGACCACGTCGACTAG-En-3'. The length of the 3' dangling-ends (En) was n = 13 or 22 bases. The identities of loop bases at positions 2 and 4 were varied. Biotinylation was varied at loop base positions 2, 3 or 4. Melting buffers contained 25 or 115 mM Na+. Average tm values for all molecules were 73.5 and 84.0°C in 25 and 115 mM Na+, respectively. Average two-state parameters evaluated from van’t Hoff analysis of the melting curve shapes in 25 mM Na+ were {Delta}HvH = 84.8 ± 15.5 kcal/mol, {Delta}SvH = 244.8 ± 45.0 cal/K·mol and {Delta}GvH = 11.9 ± 2.1 kcal/mol. In 115 mM Na+, two-state parameters were not very different at {Delta}HvH = 80.42 ± 12.74 kcal/mol, {Delta}SvH = 225.24 ± 35.88 cal/K·mol and {Delta}GvH = 13.3 ± 2.0 kcal/mol. Differential scanning calorimetry (DSC) was performed to test the validity of the two-state assumption and evaluated van’t Hoff parameters. Thermodynamic parameters from DSC measurements (within experimental error) agreed with van’t Hoff parameters, consistent with a two-state process. Overall, dangling-end DNA hairpin stabilities are not affected by dangling-end length, loop biotinylation or sequence and vary uniformly with [Na+]. Consider able freedom is afforded when designing DNA hairpins as probes in nucleic acid based detection assays, such as microarrays.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Combinatorial multiplex approaches to nucleic acid analysis are finding widespread applications and emerging as ever more important to modern methods of nucleic acid detection, discrimination and sequence analysis (1–5). Many of these methods often rely on solid support bound oligonucleotides serving as probes to hybridize with desired target DNA sequences (6–8). Many applications involve the use of high-density DNA oligonucleotide array systems to carry out hybridization reactions. Effective attachment of linear DNA probes to a solid support surface requires consideration of several factors. First, appropriate design of probe sequences and practical and effective means of fixing probes to array surfaces are required. Surfaces should not interfere with, and preferably enhance, hybridization of the proper probes with desired targets. Another important factor is the distance linear probes are raised off the surface. A recent study showed that the length of a spacer attached to linear probes and to the surface had a very dramatic effect on hybridization performance (9). Optimal spacer length was shown to be at least 40 atoms, regardless of the type of chemistry employed to attach the probes to the surface.

In addition to linear probe molecules other forms of linear DNA, i.e. dangling-ended hairpins, can offer a number of advantages for target capture in oligonucleotide hybridization based array systems. However, only a limited number of studies have been conducted to examine alternatives to linear oligonucleotide probe constructs and their effects on hybridization (10–14). With their duplex stems and loops, hairpins provide an alternative means for raising the probe sequences (via the dangling-ends) off the solid support surface. Also, it has been shown that single strands on the end of a duplex offer stabilizing advantages over their linear single strands in hybridization reactions when complexed with a single-strand target.

Recently, we reported results that showed in solution that probe/target complex formation between the dangling-end single strand and a single-strand target is significantly enhanced when a nicked duplex is formed (12–15). In thermodynamic terms, hybridization was enhanced by ~2 kcal/mol. Loops of those DNA hairpins had four T bases at positions 1,2 and 4,5. Base 3 of the loop was biotinylated uracil. Biotin in the loop allowed for affixation to a standard stryrifine microtiter plate. Subsequently, it was demonstrated that dangling-ended hairpins hybridize with linear probes at higher rates with greater association equilibrium constants than linear single-strand probes (15).

To exploit the benefits of hairpin capture probes in nucleic acid based diagnostic assays, such as multiplex hybridization assays or gene expression monitoring microarrays, requires that essential structural and sequence dependent features of the probe molecules be characterized. Toward this end, the effects of single-strand dangling-end length, biotinylation position of loop bases and loop sequence identity on the thermal stability of DNA hairpin probes have been determined and are reported here. Hairpin probes that were studied have a 16 bp duplex stem. Their melting curves were measured in 25 and 115 mM Na+ solvent environments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Thirteen DNA hairpins were synthetically prepared. These molecular constructs are shown in Figure 1A. All have the same 16 bp duplex stem, a five base single-strand loop and an n base 3' dangling-end. Hairpins were formed from the sequence, 5'-C T A G T C G A C G T G G T C C-N5-G G A C C A C G T C G A C T A G-En-3'. Lengths of the 3' dangling-end tails (En) were either n = 13 or 22 bases. These lengths were chosen for applications in other assays. Sequences of the dangling-ends are shown in Figure 1A. The bases of the loop also varied. A single biotinylated uracil base was substituted for T at positions 2, 3 or 4. Figure 1B depicts the structures of the hairpins. Thymine residues that were replaced by biotinylated uracil bases in the loops of some molecules are circled in Figure 1B.



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  		Figure 1. Hairpin probe molecules. (A) Basic design features are shown. Each hairpin had the 16 bp duplex stem shown, single-strand loop comprised of five base residues, N1 through N5, and in some cases a 3' dangling-end. Loop bases were biotinylated at either positions N2, N3 or N4. T residues at loop positions N2 and N4 were also substituted by C and G bases. (B) Structures of the 13 hairpins are shown along with their reference number. Each hairpin is numbered (1–13) corresponding to the particular modifications as shown across the bottom. Hairpin 1 is the control. Biotinylation of a loop base is denoted by a circled T residue that is replaced by a biotinylated uracil base (UB) during strand synthesis. Dangling-ends are shown to be either 13 or 22 bases long. Identities of each loop base are shown.

 
DNA strands were synthesized on an Applied Biosystems 380B automated DNA synthesizer using standard phosphoramidite chemistry (16). Biotinylated uridine phosphoramidites (Glenn Research) were incorporated into the synthesis protocol in the desired positions. Strands were deprotected, purified, desalted and vacuum dried on a speed vac concentrator and stored at –20°C (17). Strand concentrations were determined using extinction coefficients at 260 nm estimated by the nearest neighbor method (18). Sample purity and fidelity were confirmed by polyacrylamide gel electrophoresis (17).

Optical melting experiments were performed on an HP8452A single beam diode array spectrophotometer. Samples were rehydrated in melting buffer (10 mM sodium phosphate, 1 mM Na2EDTA, pH 6.8) containing either 10 or 100 mM NaCl, resulting in a total [Na+] of 25 or 115 mM. DNA samples were prepared for melting experiments as previously described (12,17). Each melting experiment contained ~0.5 OD/ml of sample in ~1.2 ml. For each sample, in both Na+ environments, at least four forward and reverse curves were collected at 268 nm, at a heating and cooling rate of 35°C/h.

Absorbance versus temperature curves were normalized to upper and lower linear base lines to obtain the fraction of broken base pairs ({theta}B) versus temperature, T curves (19,20). Derivatives of these curves yielded d({theta}B) / dT versus temperature curves. Graphical evaluation of the transition thermodynamics was performed assuming the hairpin melting transitions are two-state (21). Experimental parameters that are used in the van’t Hoff analysis are the peak height maximum on derivative curves, d({theta}B) / dTmax, and transition temperature, Tm (K), the temperature at the maximum peak height. The pertinent familiar expressions for the transition enthalpy and entropy are:

{Delta}HvH = 4RTm2[d({theta}B) / dTmax]1a

{Delta}SvH = {Delta}HvH / Tm = 4RTm[d({theta}B) / dTmax]1b

Obviously in this analysis, {Delta}HvH and {Delta}SvH are linked directly through the Tm. The van’t Hoff free energy was determined at T = 298.15 K by:

{Delta}GvH = {Delta}HvH – (T){Delta}SvH1c

Errors on these parameters were estimated from the experimental standard deviations of Tm and d({theta}B) / dTmax values.

Differential scanning calorimetry (DSC)
Measurements of the excess heat capacity, {Delta}Cpex, versus temperature, were made using a Nano II DSC instrument (Calorimetric Sciences Corp.). Procedures for sample preparation, data collection and analysis were essentially those described previously (22,23). Briefly, buffer/buffer reference and DNA/buffer sample scans were collected. Reference curves were subtracted from sample curves and corrected for total DNA concentration to yield the change in molar excess heat capacity versus temperature curves. Baselines were fit to these curves as described (24). For DSC experiments total strand concentrations ranged from 30 to 50 µM. Fitted baselines were subtracted and the DSC enthalpy, {Delta}HDSC, was determined by integration of the {Delta}Cpex versus temperature curve:

{Delta}HDSC = {int} {Delta}Cpex dT2a

The DSC entropy, {Delta}SDSC, was determined by dividing the excess heat capacity by the temperature and integrating:

{Delta}SDSC = {int} ({Delta}Cpex / T) dT2b

The DSC free energy, {Delta}GDSC, was determined at T = 298.15 K by:

{Delta}GDSC = {Delta}HDSC – (T){Delta}SDSC2c


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The results of melting experiments on the hairpins are summarized in Figure 2. The hairpin number on the horizontal axis refers to the number of the corresponding structure shown in Figure 1B. Hairpin 1 is the control (i.e. 16 bp duplex stem, no dangling-end, T5 loop). Effects of substitution and position of the biotinylated deoxyuracil, UB, for a T in the loop region were investigated using hairpins 2, 3 and 4. Like the control, these molecules did not have 3' dangling-ends. Hairpin 2 has a T-U-T3 loop, hairpin 3 has a T2-U-T2 loop, and hairpin 4 has a T3-U-T loop. The effects of substituting C and G bases for T bases at positions 2 and 4 in the loop region of a dangling-ended hairpin were investigated using hairpins 5, 6 and 7. Each hairpin has the 16 bp duplex stem of the control and a 13 base 3' dangling-end. Hairpin 5 has a T2-UB-T2 loop. Hair pins 6 and 7 have the same duplex stem with a T-C-UB-C-T loop (hairpin 6) or T-G-UB-G-T loop (hairpin 7). Hairpins 8, 9 and 10 are the same as hairpins 5, 6 and 7 without UB substituted for T in the loop. The effects of dangling-end length were investigated using hairpins 11, 12 and 13. Hairpin 11 is a longer dangling-ended analog of hairpin 5. Both have the same base pair stem, loop and 3' dangling-end sequence. The only difference between them is the length of the dangling-end, which is increased to 22 bases in hairpin 11. Hairpin 12 is the longer dangling-ended analog of hairpin 6. Again, the only difference is the 22 base dangling-end of hairpin 12. Similarly, hairpin 13 is the longer dangling-end analog of hairpin 7.



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  		Figure 2. Melting temperatures and two-state thermodynamic parameters for the 13 hairpins of this study. (A) Values of the tm recorded for hairpins 1–13 in 25 (filled circles) and 115 mM Na+ (open circles) versus the corresponding hairpin number from Figure 1A. Average melting temperatures in 25 (filled squares) and 115 mM Na+ (open squares) are denoted on the left axis. Dashed and dotted lines denote the upper and lower range of the average tm values in 25 and 115 mM Na+, respectively. (B) Values of the two-state van’t Hoff enthalpy, {Delta}HvH, for each hairpin. Symbols are the same as in (A). (C) Values of the two-state van’t Hoff entropy values, {Delta}SvH, for each hairpin. Symbols are the same as in (A) and (B). (D) Values of the van’t Hoff free energies, {Delta}GvH, for each hairpin. Symbols are the same as in (A)–(C). Average two-state thermodynamic parameters in 25 (filled squares) and 115 mM Na+ (open squares) are denoted on the left axis for each plot. Dashed and dotted lines denote the upper and lower range of the average two-state thermodynamic parameters in 25 and 115 mM Na+, respectively. Melting results reveal that all modifications examined do not significantly affect the hairpins’ stability.

 
The experimentally acquired average melting temperature value, tm, for each hairpin obtained in 25 mM Na+ (filled symbols) and 115 mM Na+ (open symbols) is plotted versus hairpin number in Figure 2A. The average over all tm values at each [Na+] is given by the filled squares on the vertical axis. Average tm values were 73.5 and 84.0°C, in 25 and 115 mM Na+, respectively. Broken lines on the plot denote the range spanned by the standard deviations from the average tm values. As can be seen, the tm values for all hairpins agreed within ±1°C in 25 mM Na+ and within ±0.5°C in 115 mM Na+. Values of the van’t Hoff enthalpy, entropy and free energy evaluated from the melting curve shapes and tm values are plotted versus hairpin number in Figure 2B–D. The reliability and quantitative accuracy of these parameter values are dictated, respectively, by the accuracy of the underlying two-state assumption. As observed for the tm values there is also reasonably good agreement between the two-state thermodynamic parameters evaluated for most of the hairpins in both Na+ environments. In 25 mM Na+, averages were {Delta}HvH = 84.8 ± 15.5 kcal/mol, of {Delta}SvH = 244.8 ± 45.0 cal/K·mol and {Delta}GvH (25°C) = 11.9 ± 2.1 kcal/mol. In 115 mM Na+ the averages were essentially the same within the standard deviations at {Delta}HvH = 80.4 ± 12.7 kcal/mol, {Delta}SvH = 225.2 ± 35.9 cal/K·mol and {Delta}GvH = 13.3 ± 2.0 kcal/mol. These results reveal that the value of {Delta}HvH is apparently independent of ionic strength, consistent with previous reports (25). Dashed and dotted lines in Figure 2B–D denote these average values for the respective parameters in 25 and 115 mM Na+. As can be seen, the two-state parameters vary only slightly from the average in both salt environments. Outliers are hairpins 2 and 4 in both salts, hairpin 1 in 25 mM and hairpin 11 in 115 mM Na+. The two-state parameters for the hairpins having UB substituted for T at different locations in the loop (hairpins 2, 3 and 4 in Fig. 1B) deviate the most from the averages and could result from greater departures from two-state melting for these molecules. As reflected in the measured tm values and largely agreeable two-state thermodynamic parameters, various modifications of the hairpins apparently do not significantly alter hairpin stability.

The results in Figure 2 also reveal several additional interesting subtle features of the hairpins. Comparisons of the melting parameters for hairpins 5, 6 and 7 with those for hairpins 11, 12 and 13, respectively, reveal there is no significance difference stability of the hairpin with dangling-end loops of 13 or 22 bases. This is consistent with published results that indicate the identity of the first base of a dangling-end is the most influential on the stability of the adjoining hairpin stem (26,27). In addition, {Delta}G is the same for hairpin 7 in both ionic strength environments. Furthermore, {Delta}G for hairpin 5 is more stable in 115 mM than in 25 mM Na+. In contrast, for the longer dangling-ended analog, hairpin 11, {Delta}G is more stable in 25 mM than in 115 mM Na+. In addition, although the tm values, {Delta}H and {Delta}S, are in reasonable agreement (with the few exceptions noted above), differences in {Delta}G are relatively larger. Although these are curious observations, most of which can probably be attributed to slight differences in deviations from two-state melting behavior, further investigations beyond the scope of this paper will be required to fully explain these results.

Direct measures of the thermodynamic parameters were also obtained by DSC in 115 mM Na+. Two hairpins (1 and 10 as denoted in Fig. 1B), were chosen because hairpin 1 showed the greatest deviation from the average thermodynamic parameters and hairpin 10 contained design factors that might be expected to affect two-state behavior. Recall, hairpin 1 is the control with a T5 loop and blunt end. Hairpin 10 contained a T-C-T-G-T loop. Hairpin 10 had a dangling-end, En, n = 13. The results from DSC measurements are shown in Figure 3. Plots of {Delta}Cpex versus temperature are shown for hairpin 1 (open circles) and hairpin 10 (closed circles). As expected, the tm values at 85.8 and 84.7°C for hairpins 1 and 10, respectively, were very similar to those measured in optical melting experiments. These values are in reasonable agreement with the average of 84 ± 0.5°C obtained from optical melts. Since melting of the hairpins is presumably a unimolecular process, concentration dependence was not expected (or observed) for the hairpins present at the 10–15 times higher concentrations required for DSC experiments. Thermodynamic parameters determined from DSC were in reasonable agreement with those evaluated from analysis of the optical melting curves. The DSC enthalpies were 92.1 and 92.6 kcal/mol for hairpins 1 and 10, respectively, compared with the average of 80.4 ± 12.7 kcal/mol evaluated in optical melting experiments. The DSC measured entropies were 256 and 259 cal/K·mol for probe molecules 1 and 10, respectively, compared with 225.2 ± 35.9 cal/K·mol from optical experiments. The DSC determined free energies were 15.8 and 15.4 kcal/mol for hairpins 1 and 10, respectively, compared with 13.3 ± 2 kcal/mol.



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  		Figure 3. DSC melting curves for selected hairpin probe molecules. Excess heat capacity, {Delta}Cp, versus temperature curves were collected for hairpin 1 (open circle) and hairpin 10 (filled circle) from Figure 1B in 115 mM Na+. Thermodynamic parameters determined from these curves are shown in the text and agreed with those obtained from two-state graphical analysis of optical melting curves.

 
The stability of the hairpins was also calculated using the M-fold program (http:/bioinfo.math.rpi.edu/~mfold/dna/ form1.cgi and included references) (data not shown). We found that the tm values and {Delta}G values calculated by M-fold were in good agreement (within error) with the values given in Figure 2. However, {Delta}H and {Delta}S from M-fold were significantly different to the values in Figure 2. This may suggest that the model in M-fold might need improvement.

Hairpins with dangling-ends can be attached to a solid phase support system and employed to capture, via hybridization, single-strand DNA or RNA target strands out of solution. Previously, we have shown that dangling-end hairpin probes capture linear single-strand targets with much better accuracy and efficiency than linear single-strand probes and prove to be superior capture moieties compared with linear single strands (12–15). Hence, hairpins should find many useful applications in oligonucleotide based array design and optimization. For these applications, it is important to understand the effects of modifications on hairpin stability. Prior to this study it was not known what effects hairpin loop sequence changes and the presence of biotinylated bases or single-strand tail length would have on the stability of DNA capture hairpins. Our results indicate that such modifications do not significantly perturb the stability of DNA hairpins in 25 or 115 mM Na+.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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