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1.  Position dependent mismatch discrimination on DNA microarrays – experiments and model 
BMC Bioinformatics  2008;9:509.
Background
The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.
Results
We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results.
Conclusion
Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.
doi:10.1186/1471-2105-9-509
PMCID: PMC2661940  PMID: 19046422
2.  Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges 
BMC Biotechnology  2008;8:48.
Background
The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations.
Results
We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form).
Conclusion
We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role.
doi:10.1186/1472-6750-8-48
PMCID: PMC2435543  PMID: 18477387
3.  Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start 
Nucleic Acids Research  2005;33(22):7129-7137.
The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0°C) than the wild-type double-stranded DNA (dsDNA, 26.0°C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0°C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2′-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0°C) than duplexes with the complementary ssDNAs (31.5 and 19.5°C). The excimer band observed in the ds2′-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2′-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2′-OMe-RNA.
doi:10.1093/nar/gki1019
PMCID: PMC1322271  PMID: 16377781
4.  Specificity of the Double-Stranded RNA-Binding Domain from the RNA-Activated Protein Kinase PKR for Double-Stranded RNA: Insights from Thermodynamics and Small-Angle X‑ray Scattering 
Biochemistry  2012;51(46):9312-9322.
The interferon-inducible, double-stranded (ds)RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays (EMSAs), and small angle X-ray scattering (SAXS) to dissect the thermodynamic basis for specificity of the dsRBD termed ‘p20’ for various RNAs, and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged (‘perfect’) or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (Kd~30 nM) and weaker binding to the bulged duplex (Kd~2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change of perfect dsRNA but largely straightens bulged dsRNA. These observations support the dsRBD’s ability to tightly bind only to A-form RNA and suggest that in a non-infected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs having complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.
doi:10.1021/bi300935p
PMCID: PMC3542976  PMID: 23140277
5.  Use of pteridine nucleoside analogs as hybridization probes 
Nucleic Acids Research  2004;32(7):e62.
The pteridine nucleoside analog 3-methyl isoxanthopterin (3-MI) is highly fluorescent, with a quantum yield of 0.88, and it can be synthesized as a phosphoramidite and incorporated into oligonucleotides through a deoxyribose linkage. Within an oligonucleotide, 3-MI is intimately associated with native bases and its fluorescence is variably quenched in a sequence-dependent manner. Bend ing, annealing, binding, digestion or cleavage of fluorophore-containing oligonucleotides can be detected by monitoring changes in fluorescence properties. We developed a single step method for detecting annealing of complementary DNA sequences using 3-MI-containing oligonucleotides as hybridization probes. One of the complementary strands contains the fluorophore as an insertion and when annealing occurs, the fluorophore bulges out from the double strand, resulting in increased fluorescence intensity. We have examined the sequence dependency, optimal strand length and impact of multiple fluorophores per strand in terms of brightness and impact on the annealing process. We describe the application of this technique to the detection of positive PCR products using an HIV-1 detection system. This sequence-dependent hybridization technique can result in fluorescence intensity increases of up to 27-fold. Fluorescence intensity increases are only seen upon specific binding to bulge-generating complements, removing issues of high background from non-specific binding.
doi:10.1093/nar/gnh060
PMCID: PMC407840  PMID: 15090623
6.  Impact of Bulge Loop Size on DNA Triplet Repeat Domains: Implications for DNA Repair and Expansion 
Biopolymers  2014;101(1):1-12.
Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, non-canonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype-phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction-dependent destabilization of the underlying double helix, and a self-structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self-structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes.
doi:10.1002/bip.22236
PMCID: PMC3920904  PMID: 23494673
DNA loop structure; DNA thermodynamics; DNA stability; trinucleotide-repeat diseases; calorimetry; circular dichroism (CD)
7.  Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure 
PLoS ONE  2012;7(12):e52994.
Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.
doi:10.1371/journal.pone.0052994
PMCID: PMC3532440  PMID: 23285245
8.  Solution structure of dAATAA and dAAUAA DNA bulges 
Nucleic Acids Research  2002;30(12):2669-2677.
The NMR structure analysis is described for two DNA molecules of identical stem sequences with a five base loop containing a pyrimidine, thymin or uracil, in between purines. These five unpaired nucleotides are bulged out and are known to induce a kink in the duplex structure. The dAATAA bulge DNA is kinked between the third and the fourth nucleotide. This contrasts with the previously studied dAAAAA bulge DNA where we found a kink between the fourth and fifth nucleotide. The total kinking angle is ∼104° for the dAATAA bulge. The findings were supported by electrophoretic data and fluorescence resonance energy transfer measurements of a similar DNA molecule end-labeled by suitable fluorescent dyes. For the dAAUAA bulge the NMR data result in a similar structure as reported for the dAATAA bulge with a kinking angle of ∼87°. The results are discussed in comparison with a rAAUAA RNA bulge found in a group I intron. Generally, the sequence-dependent structure of bulges is important to understand the role of DNA bulges in protein recognition.
PMCID: PMC117287  PMID: 12060684
9.  Dissecting the protein-RNA and RNA-RNA interactions in the nucleocapsid-mediated dimerization and isomerization of HIV-1 stemloop 1. 
Journal of molecular biology  2006;365(2):396-410.
The specific binding of HIV-1 nucleocapsid protein (NC) to the different forms assumed in vitro by the stemloop 1 (Lai variant) of the genome’s packaging signal has been investigated using electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The simultaneous observation of protein-RNA and RNA-RNA interactions in solution has provided direct information about the role of NC in the two-step model of RNA dimerization and isomerization. In particular, two distinct binding sites have been identified on the monomeric stemloop structure, corresponding to the apical loop and stem-bulge motifs. These sites share similar binding affinities that are intermediate between those of stemloop 3 (SL3) and the putative stemloop 4 (SL4) of the packaging signal. Binding to the apical loop, which contains the dimerization initiation site (DIS), competes directly with the annealing of self-complementary sequences to form a metastable kissing-loop (KL) dimer. In contrast, binding to the stem-bulge affects indirectly the monomer-dimer equilibrium by promoting the rearrangement of KL into the more stable extended duplex (ED) conformer. This process is mediated by the duplex-melting activity of NC, which destabilizes the intramolecular base pairs surrounding the KL stem-bulges and enables their exchange to form the inter-strand pairs that define the ED structure. In this conformer, high-affinity binding takes place at stem-bulge sites that are identical to those present in the monomeric and KL forms. In this case, however, the NC-induced ‘breathing’ does not result in dissociation of the double-stranded structure because of the large number of intermolecular base pairs. The different binding modes manifested by conformer-specific mutants have shown that NC can also provide low affinity interactions with the bulged-out adenines flanking the DIS region of the ED conformer, thus supporting the hypothesis that these exposed nucleotides may constitute ‘base-grips’ for protein contacts during the late stages of the viral lifecycle.
doi:10.1016/j.jmb.2006.09.081
PMCID: PMC1847390  PMID: 17070549
RNA dimerization; RNA isomerization; NC binding; NC chaperone; mass spectrometry
10.  DNA models of trinucleotide frameshift deletions: the formation of loops and bulges at the primer–template junction 
Nucleic Acids Research  2009;37(5):1682-1689.
Although mechanisms of single-nucleotide residue deletion have been investigated, processes involved in the loss of longer nucleotide sequences during DNA replication are poorly understood. Previous reports have shown that in vitro replication of a 3′-TGC TGC template sequence can result in the deletion of one 3′-TGC. We have used low-energy circular dichroism (CD) and fluorescence spectroscopy to investigate the conformations and stabilities of DNA models of the replication intermediates that may be implicated in this frameshift. Pyrrolocytosine or 2-aminopurine residues, site-specifically substituted for cytosine or adenine in the vicinity of extruded base sequences, were used as spectroscopic probes to examine local DNA conformations. An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a single-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer–template junction, showing that misalignments can be modified by neighboring DNA secondary structure. These results must be taken into account in considering the genetic and biochemical mechanisms of frameshift mutagenesis in polymerase-driven DNA replication.
doi:10.1093/nar/gkn1042
PMCID: PMC2655659  PMID: 19155277
11.  Oligonucleotides form a duplex with non-helical properties on a positively charged surface 
Nucleic Acids Research  2001;29(14):3051-3058.
The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.
PMCID: PMC55799  PMID: 11452030
12.  The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from its Structure in Crystals of 50S Ribosomal Subunits 
Biochemistry  2006;45(39):11776-11789.
Internal loops play an important role in structure and folding of RNA and in RNA recognition by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5'1009CUAAG10133'3'1168GAAGC11645' and 5'998CUAAG10023'3'1157GAAGC11535' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex, 5'1GGCUAAGAC93'3'18CCGAAGCUG105' The internal loop forms a different structure in solution than in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson–Crick UA pair.
doi:10.1021/bi0605787
PMCID: PMC4070884  PMID: 17002278
13.  DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison 
Nucleic Acids Research  2007;35(21):7197-7208.
Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters ΔH°, ΔS° and ΔG° of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson–Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.
doi:10.1093/nar/gkm865
PMCID: PMC2175334  PMID: 17947320
14.  Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics 
PLoS ONE  2012;7(8):e43862.
Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu.
doi:10.1371/journal.pone.0043862
PMCID: PMC3428302  PMID: 22952791
15.  DNA microarrays with stem–loop DNA probes: preparation and applications 
Nucleic Acids Research  2001;29(19):e92.
We have developed DNA microarrays containing stem–loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem–loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10–30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3′-deletion sets from a target and evaluated the use of stem–loop DNA arrays for detecting p53 mutations in the deletion set. The stem–loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.
PMCID: PMC60252  PMID: 11574694
16.  Cleavage of pyrene-stabilized RNA bulge loops by trans-(±)-cyclohexane-1,2-diamine 
Chemical agents that cleave HIV genome can be potentially used for anti-HIV therapy. In this report, the cleavage of the upper stem-loop region of HIV-1 TAR RNA was studied in a variety of buffers containing organic catalysts. trans-(±)-Cyclohexane-1,2-diamine was found to cleave the RNA with the highest activity (31%, 37°C, 18 h). Cleavage of the RNA in trans-(±)-cyclohexane-1,2-diamine buffer was also studied when the RNA was hybridized with complementary DNAs. A pyrene-modified C3 spacer was incorporated to the DNA strand to facilitate the formation of a RNA bulge loop in the RNA/DNA duplex. In contrast, unmodified DNAs cannot efficiently generate RNA bulge loops, regardless of the DNA sequences. The results showed that the pyrene-stablized RNA bulge loops were efficiently and site-specifically cleaved by trans-(±)-cyclohexane-1,2-diamine.
doi:10.1186/1752-153X-6-3
PMCID: PMC3319420  PMID: 22244351
17.  Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA. 
Nucleic Acids Research  1993;21(1):151-154.
Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.
Images
PMCID: PMC309077  PMID: 8441609
18.  MELTING, a flexible platform to predict the melting temperatures of nucleic acids 
BMC Bioinformatics  2012;13:101.
Background
Computing accurate nucleic acid melting temperatures has become a crucial step for the efficiency and the optimisation of numerous molecular biology techniques such as in situ hybridization, PCR, antigene targeting, and microarrays. MELTING is a free open source software which computes the enthalpy, entropy and melting temperature of nucleic acids. MELTING 4.2 was able to handle several types of hybridization such as DNA/DNA, RNA/RNA, DNA/RNA and provided corrections to melting temperatures due to the presence of sodium. The program can use either an approximative approach or a more accurate Nearest-Neighbor approach.
Results
Two new versions of the MELTING software have been released. MELTING 4.3 is a direct update of version 4.2, integrating newly available thermodynamic parameters for inosine, a modified adenine base with an universal base capacity, and incorporates a correction for magnesium. MELTING 5 is a complete reimplementation which allows much greater flexibility and extensibility. It incorporates all the thermodynamic parameters and corrections provided in MELTING 4.x and introduces a large set of thermodynamic formulae and parameters, to facilitate the calculation of melting temperatures for perfectly matching sequences, mismatches, bulge loops, CNG repeats, dangling ends, inosines, locked nucleic acids, 2-hydroxyadenines and azobenzenes. It also includes temperature corrections for monovalent ions (sodium, potassium, Tris), magnesium ions and commonly used denaturing agents such as formamide and DMSO.
Conclusions
MELTING is a useful and very flexible tool for predicting melting temperatures using approximative formulae or Nearest-Neighbor approaches, where one can select different sets of Nearest-Neighbor parameters, corrections and formulae. Both versions are freely available at http://sourceforge.net/projects/melting/and at http://www.ebi.ac.uk/compneur-srv/melting/under the terms of the GPL license.
doi:10.1186/1471-2105-13-101
PMCID: PMC3733425  PMID: 22591039
19.  Effects of polyamines on the thermal stability and formation kinetics of DNA duplexes with abnormal structure 
Nucleic Acids Research  2001;29(24):5121-5128.
The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine > spermidine > Mg2+ > Na+ > Tris–HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.
PMCID: PMC97540  PMID: 11812845
20.  Energetic signatures of single base bulges: thermodynamic consequences and biological implications 
Nucleic Acids Research  2009;38(1):97-116.
DNA bulges are biologically consequential defects that can arise from template-primer misalignments during replication and pose challenges to the cellular DNA repair machinery. Calorimetric and spectroscopic characterizations of defect-containing duplexes reveal systematic patterns of sequence-context dependent bulge-induced destabilizations. These distinguishing energetic signatures are manifest in three coupled characteristics, namely: the magnitude of the bulge-induced duplex destabilization (ΔΔGBulge); the thermodynamic origins of ΔΔGBulge (i.e. enthalpic versus entropic); and, the cooperativity of the duplex melting transition (i.e. two-state versus non-two state). We find moderately destabilized duplexes undergo two-state dissociation and exhibit ΔΔGBulge values consistent with localized, nearest neighbor perturbations arising from unfavorable entropic contributions. Conversely, strongly destabilized duplexes melt in a non-two-state manner and exhibit ΔΔGBulge values consistent with perturbations exceeding nearest-neighbor expectations that are enthalpic in origin. Significantly, our data reveal an intriguing correlation in which the energetic impact of a single bulge base centered in one strand portends the impact of the corresponding complementary bulge base embedded in the opposite strand. We discuss potential correlations between these bulge-specific differential energetic profiles and their overall biological implications in terms of DNA recognition, repair and replication.
doi:10.1093/nar/gkp1036
PMCID: PMC2800203  PMID: 19946018
21.  Parallel-stranded duplex DNA containing blocks of trans purine-purine and purine-pyrimidine base pairs. 
Nucleic Acids Research  1994;22(16):3293-3303.
A 30 base pair parallel-stranded (ps) duplex ps-L1.L2 composed of two adjoined purine-purine and purine-pyrimidine sequence blocks has been characterized thermodynamically and spectroscopically. The 5'-terminal 15 residues in both strands ('left-half') consisted of the alternating d(GA)7G sequence that forms a ps homoduplex secondary structure stabilized by d(G.G) and d(A.A) base pairs. The 3'-terminal 15 positions of the sequence ('right-half') were combinations of A and T with complementary reverse Watson-Crick d(A.T) base pairing between the two strands. The characteristics of the full length duplex were compared to those of the constituent left and right halves in order to determine the compatibility of the two ps helical forms. The thermal denaturation curves and hyperchromicity profiles of all three duplexes determined by UV absorption spectroscopy were characteristic of ps-DNA, in accordance with previous studies. The thermodynamic properties of the 30 bp duplex corresponded within experimental error to the linear combination of the two 15-mers. Thus, the Tm and delta HvH of ps-L1.L2 in 10 mM MgCl2, derived from analyses according to a statistical mechanical formulation for the helix-coil transition, were 43 degrees C and 569 kJ mol-1, compared to 21 degrees C, 315 kJ mol-1 (ps-F5.F6) and 22 degrees C, 236 kJ mol-1 (ps-GA15). The UV absorption and CD spectra of ps-L1.L2 and the individual 15-mer ps motifs were also compared quantitatively. The sums of the two constituent native spectra (left+right halves) accurately matched that of the 30 bp duplex, with only small deviations in the 195-215 nm (CD) and 220-240 nm (absorption) regions. Based on analysis by native gel electrophoresis, the sequences studied formed duplex structures exclusively; there were no indications of higher order species. Chemical modification with diethyl pyrocarbonate showed no hyperreactivity of the junctional bases, indicating a smooth transition between the two parallel-stranded conformations. We conclude that under given salt conditions, oligonucleotides with normal primary chemical structures can readily form a parallel-stranded double helix based on blocks of very disparate non-canonical purine-purine and purine-pyrimidine base pairs and without perceptible destabilization at the junction. There are biological implications of these findings in relation to genetic structure and expression.
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PMCID: PMC523721  PMID: 8078763
22.  Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability. 
Nucleic Acids Research  1990;18(21):6353-6359.
An oligodeoxyribonucleotide, d(GCTCACAAT-X-ATTGTGAGC), where X represents a hexaethylene glycol chain, was studied using circular dichroism spectroscopy. Its conformation and conformational stability were compared to those of compounds where X was replaced by four thymines and to the duplex of same sequence without loop. The compound with the hexaethylene glycol chain can form a hairpin looped structure as well as a bulged duplex structure. In both cases the duplex region of the oligodeoxyribonucleotide exhibits the same conformation. In similar conditions the oligodeoxyribonucleotide with a four thymines loop forms exclusively a hairpin structure. Comparison between the thermodynamic parameters (delta H, delta S, delta G) associated with the formation of the structure of the three compounds are presented. In the case of the compound with the hexaethylene glycol chain it is shown that the large increase in its melting temperature (by about 35 degrees C in our experimental conditions) when compared to the non looped structure is mainly due to the fact that its melting process is intramolecular (monomolecular) whereas the other one is bimolecular.
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PMCID: PMC332506  PMID: 2243780
23.  Melting studies of dangling-ended DNA hairpins: effects of end length, loop sequence and biotinylation of loop bases 
Nucleic Acids Research  2002;30(18):4088-4093.
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 ΔHvH = 84.8 ± 15.5 kcal/mol, ΔSvH = 244.8 ± 45.0 cal/K·mol and ΔGvH = 11.9 ± 2.1 kcal/mol. In 115 mM Na+, two-state parameters were not very different at ΔHvH = 80.42 ± 12.74 kcal/mol, ΔSvH = 225.24 ± 35.88 cal/K·mol and ΔGvH = 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.
PMCID: PMC137099  PMID: 12235393
24.  Hybridization of 2′-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies 
Nucleic Acids Research  2001;29(10):2163-2170.
In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2′-fluoro, 2′-O-propyl, 2′-O-methoxyethyl and 2′-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA–DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3°C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA–RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0°C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.
PMCID: PMC55455  PMID: 11353086
25.  CONFORMATIONAL EQUILIBRIA OF BULGED SITES IN DUPLEX DNA STUDIED BY EPR SPECTROSCOPY 
The journal of physical chemistry. B  2009;113(9):2664-2675.
Conformational flexibility in nucleic acids provides a basis for complex structures, binding, and signaling. One-base bulges directly neighboring single-base mismatches in nucleic acids can be present in a minimum of two distinct conformations, complicating the examination of the thermodynamics by calorimetry or UV-monitored melting techniques. To provide additional information about such structures, we demonstrate how electron paramagnetic resonance (EPR) active spin-labeled base analogues, base-specifically incorporated into the DNA, are monitors of the superposition of different bulge-mismatch conformations. EPR spectra provide information about the dynamic environments of the probe. This information is cast in terms of “dynamic signatures” that have an underlying basis in structural variations. By examining the changes in the equilibrium of the different states across a range of temperatures, the enthalpy and entropy of the interconversion among possible conformations can be determined. The DNA constructs with a single bulge neighboring a single-base mismatch (“bulge-mismatches”) may be approximately modeled as an equilibrium between two possible conformations. This structural information provides insight into the local composition of the bulge-mismatch sequences. Experiments on the bulge-mismatches show that basepairing across the helix can be understood in terms of purine and pyrimidine interactions, rather than specific bases. Measurements of the enthalpy and entropy of formation for the bulge-mismatches by differential scanning calorimetry and UV-monitored melting confirm that the formation of bulge-mismatches is in fact more complicated than a simple two-state process, consistent with the base-specific spectral data that bulge-mismatches exist in multiple conformations in the pre-melting temperature region. We find that the calculations with the nearest-neighbor (NN) model for the two likely conformations do not correlate well with the populations of structures and thermodynamic parameters inferred from the base-specific EPR dynamics probe. We report that the base-specific spin probes are able to identify a bi-stable, temperature dependent, switching between conformations for a particular complex bulged construct.
doi:10.1021/jp808260b
PMCID: PMC2902774  PMID: 19708106
Site-specific spin labeling (SDSL); extrahelical; differential scanning calorimetry (DSC); spin-lattice relaxation; thermodynamics; nearest-neighbor model

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