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Schematic representation of the apparent disparate destabilization rankings for CNC and GNG bulged duplexes as a function of base identity and flanking residues. The eight heteroduplexes are arranged/ranked according to bulge base identity (A, T, C and G appear from top to bottom) and flanking residues (CC and GG positioned in the left and right columns, respectively). The bulge-induced destabilization energies (ΔΔG in kcal/mol) are expressed relative to the parent dodecamer with the respective heteroduplexes color-coded according to their corresponding category: Red Boxes represent highly destabilized bulges that exhibit non-two-state melting behavior, whereas Blue Boxes comprise moderately destabilizing bulges that undergo ideal two-state dissociation. The magnitude of energetic destabilization (ΔΔG) may be enhanced when a specific bulge base is positioned between Gs (A and C) or within Cs (T and G) as viewed in the horizontal comparisons. Moreover, the trends observed for CNC versus GNG bulges are quite distinct in terms of the overall ΔΔG magnitudes as noted in the vertical comparisons.
Source publication
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...
Contexts in source publication
Context 1
... analysis of single base bulges embedded within a common host duplex reveals that it is difficult to define in general/ predictive terms the origins of bulge-induced thermo- dynamic and extrathermodynamic influences. This challenge is apparent when evaluating the impact of 'inserting' a base within either the sense or anti-sense strand of the parent GG/CC dodecamer, as illustrated in Figure 5. Inspection of the rows in Figure 5 reveals that the magnitude of destabilization observed for a particular bulge may be greater when the unpaired base is either positioned between Gs (i.e. ...
Context 2
... analysis of single base bulges embedded within a common host duplex reveals that it is difficult to define in general/ predictive terms the origins of bulge-induced thermo- dynamic and extrathermodynamic influences. This challenge is apparent when evaluating the impact of 'inserting' a base within either the sense or anti-sense strand of the parent GG/CC dodecamer, as illustrated in Figure 5. Inspection of the rows in Figure 5 reveals that the magnitude of destabilization observed for a particular bulge may be greater when the unpaired base is either positioned between Gs (i.e. A and C) or between Cs (i.e. ...
Context 3
... and G). Moreover, the ÁÁG ranking for a particular bulge base embedded within the CNC and GN 0 G contexts are quite distinct, as evidenced when comparing the respective columns in Figure 5. In the section that follows, we propose an alternative analysis to explore the potential origins of bulge-induced energetic perturbations that may be rationalized in terms of a 'con- certed/coupled' dependence on both base and flanking residue identity. ...
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Remdesivir (RDV) emerged as an effective drug against the SARS‐CoV‐2 virus pandemic. One of the crucial steps in the mechanism of action of RDV is its incorporation into the growing RNA strand. RDV, an adenosine analogue, forms Watson‐Crick (WC) type hydrogen bonds with uridine in the complementary strand and the strength of this interaction will c...
Citations
... These efforts have complemented biophysical studies of canonical DNA with detailed thermodynamic analysis of lesion impacts on duplex energetics [31][32][33][34][35][36][37][38][39][40][41][42] as well as lesion recognition and repair by DNA glycosylases [23,29,43,44]. While establishing the foundation required for understanding the energetic basis of complex biological systems, the Breslauer research group has focused on gene transcription regulation, repair, and replication [29,30,39,45,46]. Professor Breslauer's impressive academic career spanning five decades is chronicled in a recent retrospective [23] as his research group continues to pursue effective therapeutic interventions for combating and treating infectious diseases such as the SAR-CoV-2 pandemic [47]. ...
Drug discovery strategies have advanced significantly towards prioritizing target selectivity to achieve the longstanding goal of identifying “magic bullets” amongst thousands of chemical molecules screened for therapeutic efficacy. A myriad of emerging and existing health threats, including the SARS-CoV-2 pandemic, alarming increase in bacterial resistance, and potentially fatal chronic ailments, such as cancer, cardiovascular disease, and neurodegeneration, have incentivized the discovery of novel therapeutics in treatment regimens. The design, development, and optimization of lead compounds represent an arduous and time-consuming process that necessitates the assessment of specific criteria and metrics derived via multidisciplinary approaches incorporating functional, structural, and energetic properties. The present review focuses on specific methodologies and technologies aimed at advancing drug development with particular emphasis on the role of thermodynamics in elucidating the underlying forces governing ligand–target interaction selectivity and specificity. In the pursuit of novel therapeutics, isothermal titration calorimetry (ITC) has been utilized extensively over the past two decades to bolster drug discovery efforts, yielding information-rich thermodynamic binding signatures. A wealth of studies recognizes the need for mining thermodynamic databases to critically examine and evaluate prospective drug candidates on the basis of available metrics. The ultimate power and utility of thermodynamics within drug discovery strategies reside in the characterization and comparison of intrinsic binding signatures that facilitate the elucidation of structural–energetic correlations which assist in lead compound identification and optimization to improve overall therapeutic efficacy.
... Over the past several decades, the Breslauer, Grollman, and Johnson research laboratories have pursued a collaborative synergistic program to elucidate specific structure-function-energetic correlations in systems of biomedical relevance. This comprehensive multiparametric strategy has enabled us to characterize the overall impact of carcinogenic and mutagenic DNA lesions [1][2][3][4][5][6][7][8][9] on nucleic acid recognition [10], replication [11], and repair [10,12] (as reviewed in [13]). Applying the methodology developed and Life 2022, 12, 872 2 of 28 refined during this timeframe towards identifying effective treatment protocols to combat infectious diseases, our laboratories are currently exploring investigational compounds and repurposed molecules to assess their overall therapeutic potential as anti-SARS-CoV-2 inhibitors. ...
In an effort to identify functional-energetic correlations leading to the development of efficient anti-SARS-CoV-2 therapeutic agents, we have designed synthetic analogs of aurintricarboxylic acid (ATA), a heterogeneous polymeric mixture of structurally related linear homologs known to exhibit a host of biological properties, including antiviral activity. These derivatives are evaluated for their ability to interact with a plasma transporter protein (human serum albumin), eukaryotic (yeast) ribosomes, and a SARS-CoV-2 target, the RNA-dependent RNA polymerase (RdRp). The resultant data are critical for characterizing drug distribution, bioavailability, and effective inhibition of host and viral targets. Promising lead compounds are selected on the basis of their binding energetics which have been characterized and correlated with functional activities as assessed by inhibition of RNA replication and protein synthesis. Our results reveal that the activity of heterogeneous ATA is mimicked by linear compounds of defined molecular weight, with a dichlorohexamer salicylic-acid derivative exhibiting the highest potency. These findings are instrumental for optimizing the design of structurally defined ATA analogs that fulfill the requirements of an antiviral drug with respect to bioavailability, homogeneity, and potency, thereby expanding the arsenal of therapeutic regimens that are currently available to address the urgent need for effective SARS-CoV-2 treatment strategies.
... These energy databases also characterize DNA structures that contain biologically consequential mismatches (37,38), modified bases/lesions (37,(39)(40)(41)(42)(43)(44)(45)(46)(47), as well as nonduplex secondary structures, including the energetics of triplexes (48)(49)(50)(51)(52), tetraplexes (53-56) (associated with modulating telomere stability and epigenetic modifications) (57,58), and higher order, functionally relevant DNA assemblies (59)(60)(61)(62)(63)(64). My laboratory's triplex DNA studies were performed in collaboration with Peter Dervan's group, whom I knew well from our time together as graduate students at Yale. ...
... These energy databases also characterize DNA structures that contain biologically consequential mismatches (37,38), modified bases/lesions (37,(39)(40)(41)(42)(43)(44)(45)(46)(47), as well as nonduplex secondary structures, including the energetics of triplexes (48)(49)(50)(51)(52), tetraplexes (53-56) (associated with modulating telomere stability and epigenetic modifications) (57,58), and higher order, functionally relevant DNA assemblies (59)(60)(61)(62)(63)(64). My laboratory's triplex DNA studies were performed in collaboration with Peter Dervan's group, whom I knew well from our time together as graduate students at Yale. ...
... The examples in Figure 1 underscore the need for comparative structural studies in parallel to the corresponding energy characterizations. Furthermore, the magnitudes of these structurally/conformationally "silent," lesion-induced energy perturbations were consistent with their destabilizing impact beyond the lesion site to create domains of genome instability (37,39,40,42,78). We proposed that such energetically perturbed domains may serve as initial DNA recognition elements for the machinery of repair, a phenomenon we referred to as energetic recognition (79). ...
My personal and professional journeys have been far from predictable based on my early childhood. Owing to a range of serendipitous influences, I miraculously transitioned from a rebellious, apathetic teenage street urchin who did poorly in school to a highly motivated, disciplined, and ambitious academic honors student. I was the proverbial “late bloomer.” Ultimately, I earned my PhD in biophysical chemistry at Yale, followed by a postdoc fellowship at Berkeley. These two meccas of thermodynamics, coupled with my deep fascination with biology, instilled in me a passion to pursue an academic career focused on mapping the energy landscapes of biological systems. I viewed differential energetics as the language of molecular communication that would dictate and control biological structures, as well as modulate the modes of action associated with biological functions. I wanted to be a “molecular linguist.” For the next 50 years, my group and I used a combination of spectroscopic and calorimetric techniques to characterize the energy profiles of the polymorphic conformational space of DNA molecules, their differential ligand-binding properties, and the energy landscapes associated with mutagenic DNA damage recognition, repair, and replication. As elaborated below, the resultant energy databases have enabled the development of quantitative molecular biology through the rational design of primers, probes, and arrays for diagnostic, therapeutic, and molecular-profiling protocols, which collectively have contributed to a myriad of biomedical assays. Such profiling is further justified by yielding unique energy-based insights that complement and expand elegant, structure-based understandings of biological processes.
... Another peculiarity of mtDNA repeats is their quite short length, with majority of them shorter than 20 bases [1, 3, 5-7, 10, 20, 21]. Also, from the plethora of experimental DNAseq and/or RNAseq data as well as from the features of miRNA/mRNA interaction [69][70][71][72][73][74][75][76][77] it is known that there is a limit on the minimal length of DNA or RNA stretches that are useful for base-pairing (for example, perfect base pairing of 7-8 nucleotides of miRNA seed region is required for proper miRNA/mRNA interaction [69]). Thus, it is important to design an algorithm focused on short repeats -not all existed algorithms are able to work with imperfect repeats as short as 10 b.p. ...
... We allowed only non-tandem mismatches because of 1) the average length of previously known mitochondrial imperfect repeats is not more than 20 bases [1, 3, 5-7, 10, 20, 21]; 2) thermodynamics of duplex formation in the case of interspersed mismatches is additive, linear and wellestablished [74,75]; 3) the dependence of the duplex stability of tandem mismatch on the identity, length and context-specificity of the flanking base pairs [76]. Additionally, we keep off indels due to the same reasons (context-specific dependence of the bulges stability) [74,77]. These simplifications are biologically exceptionally meaningful due to short nature of mitochondrial imperfect repeats found in this study (on average 12 bases). ...
Background:
Mitochondria is a powerhouse of all eukaryotic cells that have its own circular DNA (mtDNA) encoding various RNAs and proteins. Somatic perturbations of mtDNA are accumulating with age thus it is of great importance to uncover the main sources of mtDNA instability. Recent analyses demonstrated that somatic mtDNA deletions depend on imperfect repeats of various nature between distant mtDNA segments. However, till now there are no comprehensive databases annotating all types of imperfect repeats in numerous species with sequenced complete mitochondrial genome as well as there are no algorithms capable to call all types of imperfect repeats in circular mtDNA.
Results:
We implemented naïve algorithm of pattern recognition by analogy to standard dot-plot construction procedures allowing us to find both perfect and imperfect repeats of four main types: direct, inverted, mirror and complementary. Our algorithm is adapted to specific characteristics of mtDNA such as circularity and an excess of short repeats - it calls imperfect repeats starting from the length of 10 b.p. We constructed interactive web available database ImtRDB depositing perfect and imperfect repeats positions in mtDNAs of more than 3500 Vertebrate species. Additional tools, such as visualization of repeats within a genome, comparison of repeat densities among different genomes and a possibility to download all results make this database useful for many biologists. Our first analyses of the database demonstrated that mtDNA imperfect repeats (i) are usually short; (ii) associated with unfolded DNA structures; (iii) four types of repeats positively correlate with each other forming two equivalent pairs: direct and mirror versus inverted and complementary, with identical nucleotide content and similar distribution between species; (iv) abundance of repeats is negatively associated with GC content; (v) dinucleotides GC versus CG are overrepresented on light chain of mtDNA covered by repeats.
Conclusions:
ImtRDB is available at http://bioinfodbs.kantiana.ru/ImtRDB/ . It is accompanied by the software calling all types of interspersed repeats with different level of degeneracy in circular DNA. This database and software can become a very useful tool in various areas of mitochondrial and chloroplast DNA research.
... 27 This follows similar conclusions for DNA bulges. 20,28 Melting temperatures for single bulges in RNA hairpins [29][30][31] as well as for longer bulges 32 further highlight these shortcommings. Therefore, there is a motivation to go beyond the nearest- neighbor model but which are still computationally efficient to handle a large number of sequences. ...
Simple one-dimensional DNA or RNA mesoscopic models are of interest for their computational efficiency while retaining the key elements of the molecular interactions. However, they only deal with perfectly formed DNA or RNA double helices and consider the intra-strand interactions to be the same on both strands. This makes it difficult to describe highly asymmetric structures such as bulges and loops, and for instance prevents the application of mesoscopic models to determine RNA secondary structures. Here we derived the conditions for the Peyrard-Bishop mesoscopic model to overcome these limitations and applied it to the calculation of single bulges, the smallest and simplest of these asymmetric structures. We found that these theoretical conditions can indeed be applied to any situation where stacking asymmetric needs to be considered. The full set of parameters for group~I RNA bulges was determined from experimental melting temperatures using an optimization procedure and we also calculated average opening profiles for several RNA sequences. We found that guanosine bulges show the strongest perturbation on their neighboring base pairs, considerably reducing the on-site interactions of their neighboring base pairs.
... Next, we investigated the influence of polyamine derivatives X0, X1 and X2 on duplex stability, inserted as bulges in the middle of the sequence (Table 1, entry ON7-ON9). The changes of duplex stability caused by single nucleotide bulges differ and depend on the type of flanking bases [46,47]. Incorporation of X0-X2 resulted in a lowering of melting temperature independently of polyamine residue. ...
... Yet, even three positive charges of a spermine residue do not neutralize the bulge effect as such. The observed changes of ΔG for the studied duplexes (ON7-ON9) are in the range observed for duplexes with a single bulge (ΔΔG, 2-6 kcal/mol) [46,47]. ...
The rationale for the synthesis of cationic modified nucleosides is higher expected nuclease resistance and potentially better cellular uptake due to an overall reduced negative charge based on internal charge compensation. Due to the ideal distance between cationic groups, polyamines are perfect counterions for oligodeoxyribonucleotides. We have synthesized non-nucleosidic analogues built from units that carry different diol structures instead of sugar residues and functionalized with polyamines. The non-nucleosidic analogues were attached as internal or 5′-terminal modifications in oligodeoxyribonucleotide strands. The thermodynamic studies of these polyaminooligonucleotide analogues revealed stabilizing or destabilizing effects that depend on the linker or polyamine used.
... Currently, only one study exists in which thermodynamic properties of 1-to 3-nt adenine or uracil bulge loop RNA were examined (Longfellow et al. 1990). Single-nucleotide bulge loops have been studied in both RNA and DNA constructs (Zhu and Wartell 1999;Tanaka et al. 2004;Blose et al. 2007;Minetti et al. 2010). A recent study examined the thermodynamic stability of the trinucleotide bulge loops in RNA (Murray et al. 2014). ...
... Single-base bulge loops have been extensively studied in DNA and RNA in 1 M NaCl (Tanaka et al. 2004;Blose et al. 2007;Minetti et al. 2010). Temperature-gradient gel electrophoresis has been performed on DNA and RNA single bulge loop constructs in one study where adenine and guanine 1-nt bulge loops had similar mobility for RNA and DNA constructs, implying similar structural conformations form in these constructs (Zhu and Wartell 1999). ...
... where N = number of nucleotides in the bulge, with N values measured between 1 and 5. Single-nucleotide bulge loops have been studied in DNA (LeBlanc and Morden 1991; Zhu and Wartell 1999;Minetti et al. 2010) but the data on larger bulge loops have not been systematically collected. In the LeBlanc and Morden study, the adenine and guanine single-nucleotide bulge loops DG W 37 loop parameters were 4.6 and 4.4 kcal/mol in the 5 ′ T-(Bulge)-T3 ′ helical context. ...
Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl2 or 1 M KCl. The ΔG 37 (°) loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔΔG° values relative to 1 M KCl were 0.47-2.06 kcal/mol more favorable for the RNA bulge loops. The ΔG 37 (°) loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride.
© 2015 Strom et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
... We thermodynamically characterize the Tg·N duplexes and their respective lesion-free T·N counterparts using temperature-dependent UV absorption spectroscopy and differential scanning calorimetry (DSC). This combined approach permits an assessment of the Tg impact on DNA duplex dissociation free energies, two-state melting cooperativities, and the related effective molecularities of the order-disorder transitions (26,(29)(30)(31). Comparison of the Tg-induced duplex destabilization with the corresponding lesion-free mismatch energetics allows us to resolve and define lesion-specific and sequence-dependent impacts. ...
... In this context, the current study is part of a broader program in which we are characterizing the energetics of biologically relevant lesions incorporated within defined DNA sequence constructs (26,(29)(30)(31)(34)(35)(36)(37)(38)(39)(40). We seek to establish structure-function-energy correlations that delineate the forces governing error-free and error-prone recognition, repair, and replication (41)(42)(43) of damaged DNA sites. ...
... Resolution of lesion-specific versus sequence-dependent impacts necessarily requires systematic investigations of biologically relevant lesions embedded within defined sequence constructs (26,(29)(30)(31)(34)(35)(36)(37)(38)(39). In the present study, we evaluate the impact of 5R-Tg on duplex dissociation energetics by site specifically incorporating the corresponding phosphoramidite as the central base residue into the host tridecanucleotide sequence 5'-GCGTACXCATGCG-3' that is hybridized with its complementary counterpart 5'-CGCATGNGTACGC-3', thereby generating Tg·A, Tg·G, Tg·T, and Tg·C duplexes. ...
The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (Tg) lesion on a family of deoxyoligonucleotide duplexes. Tg damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The Tg lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates Tg-induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-Tg isomer centered within the tridecanucleotide sequence 5'-GCGTACXCATGCG-3' (X = Tg or T) which is hybridized with the corresponding complementary sequence 5'-CGCATGNGTACGC-3' (N = A, G, T, C) to generate families of Tg-damaged (Tg·N) and lesion-free (T·N) duplexes. We demonstrate that the magnitude and nature of the Tg destabilizing impact is dependent on counterbase identity (i.e., A ∼ G < T < C). The observation that a Tg lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of Tg·N duplexes parallel their respective lesion-free T·N mismatch counterparts (i.e., G < T < C). Elucidation of Tg-induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The Tg-induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery. This article is protected by copyright. All rights reserved.
... In our study, the longest base pairing region of pP3 was located at the middle of the synthesized fragment whereas that of pP7 is located at the 3′ end. Aligning the synthesized fragment of pP7 with the phage P1 genome demonstrated that an extra base bulges outside, which could destabilize the base pairing structure (Smith et al., 2009;Minetti et al., 2010). The result showed the effect of transversion mutation introduction at different positions (either closer to the 5′ or 3′ region of the spacers), but the stabilities of the derivative plasmids of pP3 suggested that the number of matches was a more important determinant of interference effect. ...
The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titer in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP-binding site, which overlapped with a LeuO-binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titers in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.
... Nevertheless, it is advisable to employ complementary techniques such as optical methods to avoid the undesirable coupling of thermodynamic parameters (5). A combined calorimetric and spectroscopic approach yields an accurate estimate of the Gibbs energy ( G) and provides a more stringent test of the overall molecularity and validity of the two-state approximation (151)(152)(153). The integrated analysis substitutes the calorimetric enthalpy ( H cal ) in lieu of H vH within Equation 7.27 to derive the effective molecularity (i.e., n = n eff ) as proposed in studies of DNA duplex dissociation equilibria (5). ...
... The cooperativity of an unfolding process may be assessed experimentally employing a combination of calorimetric and non-calorimetric approaches including optical measurements. This empirical strategy has been applied to the characterization of protein (155) and DNA (5,151,153) unfolding/dissociation processes. The use of complementary techniques facilitates estimation of the effective molecularity (i.e., n eff ), a measure of the overall cooperativity within a biological system (as reviewed in Reference 5). ...
This chapter presents an overview of calorimetric methodology and its applications to study the thermodynamic properties of biological macromolecules. The narrative focuses primarily on experimental techniques that are utilized for the energetic characterization of association and dissociation processes, specifically isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The systems of interest are selected primarily on the basis of their relevance within the fields of biochemistry, bioengineering, and biotechnology. Special areas of emphasis include macromolecular association, folding, and recognition, encompassing protein—protein, protein—nucleic acid, and protein—ligand interactions.
