On chip quadruplex priming amplification for quantitative isothermal diagnostics.

Nucleic acid testing is a common technique for medical diagnostics. For example, it is used to detect HIV treatment failure by monitoring viral load levels. Quadruplex Priming Amplification (QPA) is an isothermal nucleic acid amplification technique that requires little power and few chemical reagents per assay, all features that make QPA well suited for point-of-care (POC) diagnostics. The QPA assay can be further optimized by integrating it with microfluidic devices that can automate and combine multiple reaction steps and reduce the quantity and cost of reagents per test. In this study, a real-time, exponential QPA reaction is demonstrated for the first time in a microfluidic chip, where the reaction was not inhibited and supported performance levels comparable to a commercially-available, non-microfluidics setup.

Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: implications for triplet expansion diseases.

We have embedded the hexameric triplet repeats (CAG)(6) and (CTG)(6) between two (GC)(3) domains to produce two 30-mer hairpins with the sequences d[(GC)(3)(CAG)(6)(GC)(3)] and d[(GC)(3)(CTG)(6)(GC)(3)]. This construct reduces the conformational space available to these repetitive DNA sequences. We find that the (CAG)(6) and (CTG)(6) repeats form stable, ordered, single-stranded structures. These structures are stabilized at 62 degrees C by an average enthalpy per base of 1.38 kcal.mol(-1) for the CAG triplet and 2.87 kcal.mol(-1) for the CTG triplet, while being entropically destabilized by 3.50 cal.K(-1).mol(-1) for the CAG triplet and 7.6 cal.K(-1).mol(-1) for the CTG triplet. Remarkably, these values correspond, respectively, to 1/3 (for CAG) and 2/3 (for CTG) of the enthalpy and entropy per base values associated with Watson-Crick base pairs. We show that the presence of the loop structure kinetically inhibits duplex formation from the two complementary 30-mer hairpins, even though the duplex is the thermodynamically more stable state. Duplex formation, however, does occur at elevated temperatures. We propose that this thermally induced formation of a more stable duplex results from thermal disruption of the single-stranded order, thereby allowing the complementary domains to associate (perhaps via "kissing hairpins"). Our melting profiles show that, once duplex formation has occurred, the hairpin intermediate state cannot be reformed, consistent with our interpretation of kinetically trapped hairpin structures. The duplex formed by the two complementary oligonucleotides does not have any unusual optical or thermodynamic properties. By contrast, the very stable structures formed by the individual single-stranded triplet repeat sequences are thermally and thermodynamically unusual. We discuss this stable, triplet repeat, single-stranded structure and its interconversion with duplex in terms of triplet expansion diseases.

Optical methods.

Equilibrium optical melting curves are used in this unit to determine thermodynamic parameters of nucleic acid complex formation. This section contains all of the relevant equations and a discussion of which are most appropriate to a given situation. Additionally, procedures are given for making preliminary determinations of molar extinction coefficients and for determining the number of oligonucleotides in a complex. The section on extinction coefficients is particularly essential to anyone needing to know solution concentrations.

Fluorescence energy transfer monitored competitive equilibria of nucleic acids: applications in thermodynamics and screening.

Precise thermodynamic characterization of nucleic acid complex stability is required to understand a variety of biologically significant events as well as to exploit the specific recognition capabilities of nucleic acids in biotechnology, diagnostics, and therapeutics. The development of a database of nucleic acid thermodynamics with sufficient precision to foster further developments in these areas requires new and improved measurement techniques. The combination of a competitive equilibrium titration with fluorescence energy transfer based detection provides a method for precise measurement of differences in free energy values for nucleic acid duplexes that far exceeds in precision those accessible via conventional methods. The method can be applied to detect and to characterize any deviation in a nucleic acid that alters duplex stability. Such deviations include, but are not limited to, mismatches; single nucleotide polymorphisms (SNP); chemically modified nucleotide bases, sugars or phosphates; and conformational anomalies or folding motifs, such as, loops or hairpins.

A more unified picture for the thermodynamics of nucleic acid duplex melting: a characterization by calorimetric and volumetric techniques.

We use a combination of calorimetric and volumetric techniques to detect and to characterize the thermodynamic changes that accompany helix-to-coil transitions for five polymeric nucleic acid duplexes. Our calorimetric measurements reveal that melting of the duplexes is accompanied by positive changes in heat capacity (DeltaCP) of similar magnitude, with an average DeltaCP value of 64.6 +/- 21.4 cal deg-1 mol-1. When this heat capacity value is used to compare significantly different transition enthalpies (DeltaHo) at a common reference temperature, Tref, we find DeltaHTref for duplex melting to be far less dependent on duplex type, base composition, or base sequence than previously believed on the basis of the conventional assumption of a near-zero value for DeltaCP. Similarly, our densimetric and acoustic measurements reveal that, at a given temperature, all the AT- and AU-containing duplexes studied here melt with nearly the same volume and compressibility changes. In the aggregate, our results, in conjunction with literature data, suggest a more unified picture for the thermodynamics of nucleic acid duplex melting. Specifically, when compared at a common temperature, the apparent large differences present in the literature for the transition enthalpies of different duplexes become much more compressed, and the melting of all-AT- and all-AU-containing duplexes exhibits similar volume and compressibility changes despite differences in sequence and conformation. Thus, insofar as thermodynamic properties are concerned, when comparing duplexes, the temperature under consideration is as important as, if not more important than, the duplex type, the base composition, or the base sequence. This general behavior has significant implications for our basic understanding of the forces that stabilize nucleic acid duplexes. This behavior also is of practical significance in connection with the use of thermodynamic databases for designing probes and for assessing the affinity and specificity associated with hybridization-based protocols used in a wide range of sequencing, diagnostic, and therapeutic applications.

A quantitative method for evaluating the stabilities of nucleic acids.

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed "reference" duplex (usually a Watson-Crick duplex) and a related "test" duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.

Effects of 3,N4-ethenodeoxycytidine on duplex stability and energetics.

The exocyclic cytosine adduct 3,N4-ethenocytosine is highly mutagenic in mammalian cells. We describe the impact of this adduct on DNA duplex stability. The adduct does not disrupt the overall B-form DNA structure; however, structural accommodation of the adduct is necessary at the lesion site. Despite the relatively small structural perturbation imparted by the adduct, there is a large adduct-induced destabilization of the DNA duplex. This destabilization is observed to be independent of the cross-strand partner base and neighbouring base pairs. The thermodynamic origins of the destabilization are, however, strongly dependent on the cross-strand partner base and neighbouring base pairs. Comparisons are made between the impact of the 3,N4-ethenocytosine adduct and other lesions on DNA thermodynamics. The lesions are similar in that all result in destabilization of the DNA duplex. The magnitudes and the thermodynamic origins of that destabilization vary widely, the 3,N4-ethenocytosine adduct being dramatically more destabilizing than other lesions. The impact of damaged sites on the stability of the DNA helix suggests that energetic differences between damaged and normal DNA may contribute to the recognition of damage by the cellular DNA repair machinery.

The impact of an exocyclic cytosine adduct on DNA duplex properties: significant thermodynamic consequences despite modest lesion-induced structural alterations.

The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.

Thermodynamic consequences of an abasic lesion in duplex DNA are strongly dependent on base sequence.

The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C.G or G.C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magnitude of this impact being strongly dependent on sequence context.

Influence of the oxidatively damaged adduct 8-oxodeoxyguanosine on the conformation, energetics, and thermodynamic stability of a DNA duplex.

As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic and calorimetric study on a family of DNA duplexes both with and without the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG). Specifically, we have studied a family of eight 13-mer duplexes of the form [5'-GCGTAC[G* or G]CATGCG-3'].[3'-CGCATG[C, A, T, or G]GTACGC-5'] in which G* is the 8-oxodG lesion. These eight duplexes, which we designate by the identity of the variable central base pair (e.g., G*C), reflect two subsets: four duplexes in which the modified guanine base is positioned opposite each of the four possible canonical residues (G*C, G*A, G*G, G*T) and the corresponding four "control" duplexes in which the guanine is not modified (GC, GA, GG, GT). The data derived from our spectroscopic and calorimetric measurements on these eight duplexes allow us to evaluate the influence of the 8-oxodG lesion, as well as the base opposite the lesion, on the conformation, the thermal and thermodynamic stability, and the melting thermodynamics of the host DNA duplex. We find that modification of dG to 8-oxodG (G*) does not change the global DNA duplex conformation as judged by circular dichroism spectra. Despite this structural similarity, our data reveal that the dG to dG* modification does influence duplex thermal and thermodynamic properties, some of which depend on the base opposite the lesion. Thus, apparent structural identity does not mean that two duplexes necessarily will exhibit equivalent thermal and/or thermodynamic properties. In general, we find that the thermodynamic effects induced by the lesion (e.g., GC vs G*C) or by mismatched base pairs (e.g., GC vs GG) can result in relatively large changes in enthalpy which are partially or wholly compensated entropically to produce relatively modest changes in free energy. Our data also suggest that the biologically observed differential recognition of 8-oxodG duplexes and the preferential nucleotide insertion opposite 8-oxodG residues cannot be rationalized simply in terms of large thermodynamic differences.

Calorimetry of proteins and nucleic acids.

The availability of sensitive calorimetric instrumentation has led to a considerable increase in thermodynamic studies of proteins, nucleic acids, and their interactions. This article reviews some of the recent contributions of calorimetry to characterizing the thermodynamic origins of protein and nucleic acid stability and conformational preferences, as well as the interactions of proteins with each other, with small molecules, and with nucleic acids.

Berenil [1,3-bis(4'-amidinophenyl)triazene] binding to DNA duplexes and to a RNA duplex: evidence for both intercalative and minor groove binding properties.

Berenil is an antitrypanosomal agent that binds to nucleic acid duplexes. The generally accepted mode of berenil binding is via complexation into the minor groove of AT-rich domains of DNA double helices. We find that berenil can bind to RNA as well as DNA duplexes, while exhibiting properties characteristic of both intercalation as well as minor groove binding. More specifically, we use spectroscopic, calorimetric, and hydrodynamic techniques to characterize berenil binding to four DNA duplexes and to one RNA duplex. Our results reveal the following features: (i) Berenil binding to the poly[d(A-T)]2, poly(dA).poly(dT), poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes exhibits intercalative as well as minor groove binding characteristics. (ii) The apparent "site sizes" associated with berenil binding to these five duplexes range from 1 to 13 base pairs per bound berenil and depend, in part, on the host duplex. One of the site sizes common to all five duplexes is consistent with berenil binding to the minor groove. (iii) The apparent berenil binding affinity follows the hierarchy: poly(dA).poly(dT) > poly-[d(A-T)]2 approximately poly[d(I-C)]2 >> poly(rA).poly(rU) > poly[d(G-C)]2. (iv) Viscometric data reveal properties characteristic of a significant contribution from an intercalative mode of binding when berenil interacts with the poly[d(A-T)]2, poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes, while revealing an apparent nonintercalative mode when the drug binds to the poly(dA).poly(dT) duplex. (v) Berenil binding unwinds negative supercoils in the pBR322 plasmid, an observation consistent with an intercalative mode of binding to duplex DNA. (vi) Salt-dependent melting data suggest that both positively charged amidino groups of berenil participate in the complexation of the drug to the poly[d(I-C)]2, poly[d(A-T)]2, poly(dA).poly(dT), and poly(rA).poly(rU) duplexes, while also suggesting that the binding event is site-specific. In the aggregate, our results suggest that, in contrast to the conventional wisdom, berenil can exhibit intercalative as well as minor groove binding properties when it binds to both DNA and RNA duplexes, while also exhibiting a preference for DNA duplexes with unobstructed minor grooves. We comment on the potential correlation between drugs, such as berenil, that exhibit "mixed" binding motifs and those that express anticancer activity via inhibition of topoisomerase I activity.

The thermodynamics of DNA structures that contain lesions or guanine tetrads.

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data.

Thermodynamics of an intramolecular DNA triple helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally induced structural transitions.

We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5'-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3') which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5 loop. The 3'-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5 loop, form Hoogsteen interactions in the major groove of the Watson-Crick hairpin. From ultraviolet (UV) melting studies we find that the 31-mer exhibits either one or two transitions, depending on solution conditions. We use pH- and temperature-dependent circular dichroism (CD) to assign the initial and final states associated with each transition. We find that the disruption of the Hoogsteen hairpin is accompanied by a release of protons and an uptake of sodium ions while the disruption of the Watson-Crick hairpin is accompanied by a release of sodium ions with no change in protonation state. From these data, we construct a phase diagram for this intramolecular DNA triple helix as a function of pH, sodium ion concentration, and temperature. We characterize the energetics of each transition using a van't Hoff analysis and differential scanning calorimetry (DSC). Significantly, the DSC data provide a model-independent thermodynamic characterization of the thermally induced transitions of this triplex. By combining the spectroscopic and calorimetric data, we develop a semi-empirical model which describes the state of the 31-mer as a function of pH, sodium ion concentration, and temperature. With this model we successfully predict characteristics of the 31-mer, which are beyond the data which are used in establishing the model (for example, the salt dependence of the apparent pKa of the Hoogsteen strand). This semi-empirical model may serve as a prototype for developing a method to predict the phase diagrams of intramolecular triple helix systems.

Nucleic acid hybridization: triplex stability and energetics.

In this chapter, we review the current state of the thermodynamic database for triple helical oligonucleotide hybridization reactions and present a critical assessment of the methods used to obtain the relevant data. The thermodynamic stability of triple-helix oligonucleotide constructs is discussed in terms of its dependence on temperature, chain length, pH, salt, base sequence, base and backbone modifications, and ligand binding. In particular, we examine the coupling of hybridization equilibria to proton, cation, and drug-binding equilibria. Throughout the chapter, we emphasize that a detailed understanding of the endogenous and exogenous variables that control triplex stability is required for the rational design of oligonucleotides for specific therapeutic, diagnostic, and/or biotechnological applications, as well as for elucidating the potential cellular roles of these higher-order nucleic acid complexes.

Influence of drug binding on DNA hydration: acoustic and densimetric characterizations of netropsin binding to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes and the poly(dT).poly(dA).poly(dT) triplex at 25 degrees C.

We use high-precision acoustic and densimetric techniques to determine, at 25 degrees C, the changes in volume, delta V, and adiabatic compressibility, delta Ks, that accompany the binding of netropsin to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes, as well as to the poly(dT).poly(dA).poly(dT) triplex. We find that netropsin binding to the heteropolymeric poly(dAdT).poly(dAdT) duplex is accompanied by negative changes in volume, delta V, and small positive changes in compressibility, delta Ks. By contrast, netropsin binding to the homopolymeric poly(dA).poly(dT) duplex is accompanied by large positive changes in both volume, delta V, and compressibility, delta Ks. Furthermore, netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes changes in both volume and compressibility that are nearly twice as large as those observed when netropsin binds to the poly(dA).poly(dT) duplex. We interpret these macroscopic data in terms of binding-induced microscopic changes in the hydration of the DNA structures and the drug. Specifically, we find that netropsin binding induces the release of approximately 22 waters from the hydration shell of the poly(dAdT).poly(dAdT) heteropolymeric duplex, approximately 40 waters from the hydration shell of the poly(dA).poly(dT) homopolymeric duplex, and about 53 waters from the hydration shell of the poly(dA).poly(dT), induces the release of 18 more water molecules than netropsin binding to the heteropolymeric duplex, poly(dAdT).poly(dAdT). On the basis of apparent molar volume, phi V, and apparent molar adiabatic compressibility, phi Ks, values for the initial drug-free and final drug-bound states of the two all-AT duplexes, we propose that the larger dehydration of the poly(dA).poly(dT) duplex reflects, in part, the formation of a less hydrated poly(dA).poly(dT)-netropsin complex compared with the corresponding poly(dAdT).poly(dAdT)-netropsin complex. In conjunction with our previously published entropy data [Marky, L. A., & Breslauer, K. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4359-4363], we calculate that each water of hydration released to the bulk solvent by ligand binding contributes 1.6 cal K-1 mol-1 to the entropy of binding. This value corresponds to the average difference between the partial molar entropy of water in the bulk state and water in the hydration shells of the two all-AT duplexes. When netropsin binds to the poly(dT).poly(dA).poly(dT) triplex, the changes in both volume and compressibility suggest that the binding event induces more dehydration of the triplex than of the duplex state. Specifically, we calculate that netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes the release of 13 more waters than netropsin binding to the poly(dA).poly(dT) duplex.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of base composition, base sequence, and duplex structure on DNA hydration: apparent molar volumes and apparent molar adiabatic compressibilities of synthetic and natural DNA duplexes at 25 degrees C.

Using high-precision densitometric and ultrasonic measurements, we have determined, at 25 degrees C, the apparent molar volumes, phi V, and the apparent molar compressibilities, phi KS, of five natural and three synthetic B-form DNA duplexes with varying base compositions and base sequences. We find that phi V ranges from 152.0 to 186.6 cm3 mol-1, while phi KS ranges from -73.0 x 10(-4) to -32.6 x 10(-4) cm3 mol-1 bar-1. We interpret these data in terms of DNA hydration which, by the definition employed in this work, refers to those water molecules whose density and compressibility differ from those of bulk water due to interactions with the DNA solute. This definition implies that hydration depends not just on the quantity but also on the quality of the solvent molecules perturbed by the solute. In fact, we find that the number of water molecules perturbed by the DNA duplexes (the quantity of water in their hydration shells) is approximately the same for all of the B-form double helixes studied, while the quality of this water differs as measured by its density and compressibility, thereby yielding differences in the overall hydration properties. Specifically, we find a linear relationship between the density and the coefficient of adiabatic compressibility, beta Sh, of water in the hydration shell of the DNA duplexes, with the range of values for beta Sh being only 65-80% of the value of bulk water.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of an exocyclic guanine adduct on the thermal stability, conformation, and melting thermodynamics of a DNA duplex.

As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic study on pairs of DNA duplexes both with and without an exocyclic guanine lesion. Specifically, we have studied a family of four 13-mer duplexes of the form d(CGCATGYGTACGC).d(GCGTACZCATGCG) in which Y is either the normal deoxyguanosine residue (G) or the exocyclic guanine adduct 1,N2-propanodeoxyguanosine (X), while Z is either deoxycytosine (C) or deoxyadenosine (A). Thus, the four duplexes studied, which can be designated by the identity of their central Y.Z base pair, are a Watson-Crick duplex (GC), a duplex with a central mismatch (GA), and two duplexes with exocyclic guanine lesions (X), that differ only by the base opposite the lesion (XC and XA). The data derived from our spectroscopic measurements on these four duplexes have allowed us to evaluate the influence of the exocyclic guanine lesion, as well as the base opposite the lesion, on the conformation, thermal stability, and melting energetics of the host DNA duplex. To be specific, our circular dichroism (CD) spectra show that the exocyclic guanine lesion induces alterations in the duplex structure, while our temperature-dependent optical measurements reveal that these lesion-induced structural alterations reduce the thermal stability, the transition enthalpy, and the transition free energy of the duplex.(ABSTRACT TRUNCATED AT 250 WORDS)