- Open Access
Compatible solute influence on nucleic acids: Many questions but few answers
© Kurz; licensee BioMed Central Ltd. 2008
- Received: 21 January 2008
- Accepted: 03 June 2008
- Published: 03 June 2008
Compatible solutes are small organic osmolytes including but not limited to sugars, polyols, amino acids, and their derivatives. They are compatible with cell metabolism even at molar concentrations. A variety of organisms synthesize or take up compatible solutes for adaptation to extreme environments. In addition to their protective action on whole cells, compatible solutes display significant effects on biomolecules in vitro. These include stabilization of native protein and nucleic acid structures. They are used as additives in polymerase chain reactions to increase product yield and specificity, but also in other nucleic acid and protein applications.
Interactions of compatible solutes with nucleic acids and protein-nucleic acid complexes are much less understood than the corresponding interactions of compatible solutes with proteins. Although we may begin to understand solute/nucleic acid interactions there are only few answers to the many questions we have. I summarize here the current state of knowledge and discuss possible molecular mechanisms and thermodynamics.
- Compatible Solute
- Glycine Betaine
- Nucleic Acid Basis
- Free Hydration Energy
Beneficial effects of compatible solutes on nucleic acids and nucleic acid/protein complexes are mainly known from improvements in yield and specificity of polymerase chain reaction (PCR), see [26–32] for examples. But effects also extend to nucleic acid stabilization , improvement of protein/nucleic acid complex formation , nucleic acid purification  and cell free transcription [36, 37] as well as modulation of restriction enzyme function [38, 39]. Contrary to other well known effector molecules like polyamines which stabilize negatively charged macromolecules due to their cationic nature , the mode of interaction of zwitterionic, anionic and uncharged low molecular weight compounds with nucleic acids is barely understood. There are some obvious possibilities how they might influence nucleic acids. Among them are changes in the electrostatic environment , intercalation  and a role as anti-intercalators .
In this review I will start with an overview of known effects of compatible solutes on nucleic acids, focusing on double stranded DNA since a wealth of data can be retrieved from this area of work. Still "DNA comes in many forms" , a fact we are aware of since 1957 , only four years after Watson and Crick presented their theory of the double stranded DNA helix . Therefore I am also going to present more complex structures like triple- and quadruple helices. Considering the importance of riboswitches  and the recent advances which have been made in this field  interference of small metabolites with RNA has become of prime importance. Therefore RNA interactions with small osmolytes, be they direct  or indirect, might play an essential role in regulation of compatible solute biosynthesis and adaptation. After discussing potential models for molecular mechanics of interactions, and first steps towards applying those models to nucleic acids, I will address the questions still unanswered. A few thoughts will be given towards computational methods before I draw my conclusions and give some future prospects.
Inorganic ions are not meant to be a major subject of this review. Their influence on nucleic acids, especially RNA, and their structure has been reviewed thoroughly by Draper  and newer findings, including CS effects, have been presented recently . Nevertheless, we have to consider effects of inorganic ions when interpreting experimental data since they are an indispensable component of buffers and the native environment of nucleic acids. Most obvious is their influence on DNA stability (see next section), which is reflected in those melting temperature (TM) calculations that consider ion concentrations or solvent ionic strength. The approach of Frank-Kamenetskii  may serve here as an example:
TM (°C) = 87.16 + 0.345 × (%GC) + log [Na+] × (20.17 - 0.066 × (%GC)), [Na+] given in mM, (%GC) is GC content given in a range from 0 to 1.
Therefore cations are of special interest, among them physiologically important species like bivalent magnesium and monovalent sodium and potassium. They act as counterions to the phosphate backbone  of nucleic acids and – due to their charge screening effect – reduce repulsive force between the two stands, hence the increase in DNA melting temperature. Cations are able to counteract effects of compatible solutes on nucleic acids and vice versa [25, 53]. This phenomenon is also linked to the counterion atmosphere [54, 55] and therefore to the counterion condensation theory, originally introduced by Manning  and Oosawa  and later refined by Shaughnessy .
In accordance with their native function in nucleic acid stability and functionality of nucleic acid processing enzymes, Mg2+ and K+ are able to stabilize nucleic acids in in vitro assays . But we also already know that, depending on concentrations, bivalent ion species can compact DNA and induce its aggregation . This compaction only occurs in a narrow range of concentrations. Apparently hydration of the cations, especially the size of the hydration shell, plays an important role: Li+ was reported to behave very differently from other monovalent alkali ions . Ions are also known to influence the stability of more exotic DNA structures [61, 62] and to be involved in halophilic adaptation of protein DNA interactions [63–65]. This is of importance when comparing and contrasting organisms which accumulate salt for osmoadaptation (salt-in strategy) with those using compatible solutes (CS strategy). K+ is not only used by salt-in organisms (which might have equally high Cl- concentrations inside ) but also by compatible solute producers in their early up-shift adaptation phase [67, 68]. Organisms using the CS strategy usually employ potassium as a transient solute and glutamate as its counterion. All this shows us that an in vivo model of nucleic acid/compatible solute interactions definitely has to include their ionic environment.
A first approach to compatible solute nucleic acid interactions should aim at the effect of compatible solutes on the melting of double stranded DNA (dsDNA).
Many formulas for calculation of dsDNA melting temperature TM exist. The Wallace rule  is the most simple one, but it is limited to DNA oligomers with 14 to 20 nucleotides and a highly specialized formula which requires the use of certain buffer concentrations. As already mentioned in the previous section, other methods for TM calculation include terms for counterion concentrations or ionic strength of the buffer, e.g. . Even more sophisticated models include thermodynamic lattice models and nearest neighbour calculations [70–72].
When thinking about nucleic acid stability the first thing which comes to mind is the DNA melting curve or, more precisely, thermal stability and thermal melting of dsDNA, which seems like a simple enough experiment. However, the simplicity behind this is deceiving. One is most likely tempted to forget about the influence of counterions, general ionic strength, solvent dielectricity and pH on dsDNA stability. In addition, other DNA structures and RNA are neglected completely. But even with a focus on thermal dsDNA melting the topic is quite complex [80–82] and, besides the melting point, we are able to gain thermodynamic data from a melting curve. Since even small PCR products can have complex melting profiles , the exact determination of melting temperatures might prove to be an arduous task with researchers being eager to find new efficient methods or to improve existing ones .
Thus, looking at the principles of nucleic acids stability does not really help us to find answers but points out even more factors which we have to keep in mind, as for example: the mechanics of base stacking and pairing , the main factors in DNA stability , intermediate states in DNA melting including bubble nucleation, cooperativity  and their fluctuation , local cooperativity in DNA melting  or sensitivity of TM to the presence of counterions . All of these might or might not be affected by compatible solutes.
What other factors, besides compatible solutes, are known to influence nucleic acid stability? In 1999 Spink and co-workers demonstrated that the stability of DNA duplex and triplex structures not only depends on molecular forces such as base pairing or tripling or electrostatic interactions but also on its aqueous environment . Thermal melting of RNA for example was shown to depend on pH and solvent . Recent investigations point out the influence of mixed solvents and their different dielectric constants on DNA denaturation . Such solvent-dependent effects indicate indirect effects on nucleic acid stability, possibly an influence of the solute or co-solvent on the structure of the solvent (water) or its electrochemical properties.
On the other hand, we are presented with reports on the proportionality of effects of intercalating substances on DNA duplex melting , which are quite similar to the linear dependency of solute concentrations and DNA melting points reported by Rees and co-workers . Surfactants are known to interact with DNA intercalators and, depending on their surface charge, to intercalate or to affect TM to varying degrees . SYBR®-green, commonly used in realtime PCR applications, even binds preferentially to certain DNA structures .
Again we have a huge amount of data and cannot really decide whether solutes have an indirect effect, interact directly with nucleic acids or both. To make the uncertainty complete, different compatible solutes might have different modes of action. But we are not completely left in the dark. Spink and co-workers have recently published calorimetric data and consider the importance of enthalpies of DNA melting in the presence of osmolytes .
In a polymerase chain reaction we have to take care of the proteins and their interactions with DNA in addition to thermal denaturation of the DNA template and formation of new DNA duplexes. To complicate matters the enzymatic reaction itself might be influenced by the addition of solutes or co-solvents, but we cannot ignore this aspect, especially since a lot of data concerning the influence of solutes are presently available. Additives which improve PCR reactions are highly sought after since even a simple "standard" application to amplify a certain region of DNA might not work properly with a particular sequence. Possible reasons might range from the simple, like GC-content or secondary structure formation of the template, to the obscure, like the complexity of compounds in a diagnostic PCR performed on clinical samples . Cations, mainly K+ and Na+ are among the main inhibitors of a PCR and Mg2+ – which is needed by the DNA polymerase – has a large, concentration dependent impact on PCR specificity .
It was the group of Weissensteiner which presented glycine betaine in 1996 as the first substance to counteract the effect of NaCl . They introduced the term cosolute for such additives. Ever since betaine has been used as a PCR facilitator: as a single compound [29, 97, 98], in combination with DMSO [26, 32, 99–101] or together with BSA . It has since been used on templates with varying GC content, to enhance formation of long PCR products [103, 104] in low temperature PCR with heat labile polymerases  and in diagnostic PCR . Sugars like trehalose or sucrose [107–109] have also been used with similar success as have been low molecular weight sulfones , amides  and sulfoxides . In addition, recent data demonstrate that even synthetic derivatives of ectoines can act as powerful PCR enhancers .
Most of those reports lack a reasonable explanation as to how these compounds work. Some, for example sucrose and trehalose  or sarcosine , are supposed to stabilize the DNA polymerase. Betaine and again trehalose are believed to facilitate PCR by lowering TM or eliminating its dependency on base composition , which would be consistent with the early observation that DNA regions with high TM prevent amplification . Furthermore, certain DNA sequences can cause DNA polymerase to pause, a phenomenon which is again counteracted by betaine . More exotic substances like 7-deaza-2'-deoxyguanosine compete with dNTPs for the active site and slow down PCR . This in turn gives a proofreading DNA polymerase more time to detect mismatched bases. But this is not to be confused with a true compatible solute, it is rather a PCR enhancer working on a different level.
Similar to the effects reported for nucleic acid stability and DNA melting we do not get a clear answer with respect to a possible mode of action. In spite of the large amount of reports on compatible solute effects in PCR we only get a vague picture as to what aspects of a PCR might be affected. And again we learn that different solutes react differently.
Already in 1998 Record  included DNA protein interactions into his considerations on biophysical aspects of bacterial adaptation to osmolarity. With insufficient data to get a more detailed model, low water activity as the cause for molecular crowding and reduced biopolymer diffusion and interactions was proposed as the reason behind reduced growth rates. However, observing a whole cell might not be a good idea. Nucleic acid/protein structures without catalytic function would be more ideal targets for observations of compatible solutes effects. Unfortunately, this was only done in detail for the reconstitution of functional 50S ribosomes . Though the model itself is not really simple and does not yield mechanistic information some impressive data were obtained: The authors demonstrated that, when using in vitro transcripts of Escherichia coli 23S rRNA, ribosome reconstitution was stimulated by a factor of up to 100 in the presence of trimethyleaminoxide in combination with an antibiotic. More recent but less detailed data are available for a complex formed by leucine-responsive regulatory protein (LRP) with ribosomal DNA (rDNA) . Here the CS ectoine stabilized the complex while the amino acid leucine had a destabilizing effect.
Returning to nucleic acid-protein interactions with catalytic activity I would like to draw your attention to restriction endonuclease complex formation and activity. Recent data show, that the dissociation of the EcoRI DNA complex is slowed down drastically by the neutral osmolytes glycine, glycerol, triethylene glycol and sucrose , an effect highly dependent on the resulting osmolarity and subsequent low water activity [120, 121]. In another study glycine betaine was reported to improve restriction of DNA resistant to digestion despite the presence of appropriate recognition sites . This was compared to the positive effect of betaine on PCR and contributed to the same – unknown – mechanisms. The opposite effect, protection to the point of complete inhibition of restriction, was observed for tetrahydropyrimidine derivatives and a range of type II restriction endonucleases . The latter observation can be traced back to a patent application by the group of Lapidot [122, 123]. In their work we find some NMR data which imply that proximity or binding of compatible solutes to guanine might play a role in this process. These findings are consistent with the general observation that CS have more pronounced effects on GC-rich sequences, which was made as early as 1993 . Unfortunately the Lapidot group had published only one more project concerned with the influence of osmolytes on nucleic acid/protein interaction  and, apparently, did not continue NMR studies on the topic.
The findings of Lee and Gralla have a slightly more complex background: potassium glutamate takes part in the regulation of promoters for genes involved in osmoadaptation  and can even act as a global inhibitor of housekeeping genes under osmotic stress . These contribute to changes in the DNA double helix structure, probably alteration of DNA bends, and depend on glutamate concentration. Related to this observation are data from the regulation of the betaine uptake system Bus in Lactococcus lactis . The in vitro stability of a complex of regulator BusR and promotor busA strongly depended on the ionic strength of the buffer. Unfortunately no attempt was made to explain the molecular basis.
Naturally we would like to derive a general underlying concept for the influence of solutes on promoters. In this context it might be of interest to learn that the promoter region of a vast array of Human genes was reported to have a conserved set of distinct flexible and rigid regions independent of the consensus sequence . Therefore, it would be challenging to test the influence of compatible solutes on mechanical properties of promoter regions, an aspect which so far has been neglected. A possible area worthy of investigation deals with solvent property changes, such as the effect of solvent dielectric constant on DNA torsional properties . Again dielectric constants have not been reported for CS solutions.
So far there is only one serious approach towards describing the molecular mechanisms of compatible solute action on nucleic acid properties, a fact which can be largely ascribed to the lack of basic mechanistic data. As already mentioned above, research on protein-solute interactions is more advanced and has led to a number of models. These might prove more or less useful for nucleic acids, depending on the general mode of interaction and the solute in question.
Prior to analysing those models against the background of nucleic acid research, we have to ask ourselves whether and up to which level we can treat nucleic acids and proteins alike. With a view to nucleic acid aptamers [130, 131], the complexity of structures and surfaces of nucleic acids, in many ways similar to the antigen recognition site of the antibody Fab chain, seems to be comparable to that of proteins. Therefore, one might be tempted to assume similar macromolecular properties. But what about a comparison in detail?
In both cases we have a backbone/sidechain structure. However a phosphate/sugar backbone will behave differently in comparison to a polypeptide backbone, and only four hydrophobic bases offer less sequence flexibility than the 20 proteinogenic sidechains (derivatives of both species not counted). Similarly, it would be questionable to compare double-stranded helical DNA or RNA to alpha-helical or beta-strand like structures and other conformations to omega-loops, random coils and the like. Returning to the phosphates of the nucleic acids backbone, the reason for the most striking difference becomes apparent: nucleic acids always have a strong negative net charge, even when compared to acidic proteins, and thus are surrounded by an atmosphere of positively charged countercations.
In view of all those differences, what do nucleic acids and proteins have in common? The answer lies in the basic physical principles behind interactions, which include the surrounding water as solvent and which are for both biopolymers the driving force to fold. Therefore in general all models concerning those physical principles should be applicable to proteins and nucleic acids alike.
The preferential interaction theory was phrased by Arakawa and Timasheff in 1983  following the observation that both, glycerol and betaine, besides increasing the water surface tension are excluded from protein surfaces. The theory provides good explanations for a wide range of phenomena concerning the interactions of solutes, salts and biomolecules in water (though it might be better to say, and water). A revised version of this theory is given in reviews by Timasheff 1998  and 2002  with latest results from ion-exchange chromatography studies . Recent publications also include molecular dynamics simulations showing ectoine preferential exclusion/interaction .
1) Solute molecules bind preferentially to the protein surface and, as a consequence, less water molecules bind. The protein is less hydrated and we observe a lower free hydration energy ΔGhb < ΔGh. This is indeed a similar situation as in the water replacement theory  which will be presented next.
2) Solute molecules are inert and statistically distributed within the solution and on the protein surface. Free hydration energies ΔGhi and ΔGh are the same.
3) The solute is preferentially excluded from the protein surface, equivalent to a state of preferential hydration. Total exclusion of the solute from the molecule surface would be one extreme and similar to a solution of protein in water. However this situation would only represent a similar but not an equal state, as we need to consider a thermodynamic difference in the surrounding medium, a concentration gradient of the solute from the bulk solution towards the protein. The free hydration energy ΔGhx is higher than ΔGh.
Interestingly, nearly all known stabilizing solutes are excluded from the protein surface. The stability of a protein under denaturing influences is now determined by the difference ΔΔG = ΔGhb, i or x – ΔGh. An inert solute will not have any influence at all, beside a general osmotic effect. A preferentially binding solute will destabilize the protein since ΔΔG is negative, and energy is released, which promotes unfolding of the protein. On the other hand, when solutes are excluded they have a positive ΔΔG, which means that unfolding needs additional energy and is inhibited.
The water replacement theory by Clegg and co-workers  is based on the observation that many organisms are able to lose a larger amount of cellular water and return to full activity after rehydration. Cellular structures can be protected by the accumulation of certain compatible solutes and their interactions with surfaces. Since, in this model, water is replaced by solutes, it seems to be the complete opposite of the preferential interaction theory. However, we have to take into account that these observations were made with dried samples and therefore at very high solute concentrations. Replacement therefore, appears to be a very special situation of extreme low water activity, while the interaction model is valid for the more diluted range of solute concentrations. The importance of this model lies in the fact, that the relative affinity of a solute towards water or protein may well be concentration dependent, especially when desiccation (i.e. very low water activity) is involved .
Water density fractions
The impact of macromolecules and compatible solutes on the three-dimensional structure of water hydrogen bond networks and water density fractions was first studied by Wiggins [23, 137, 138]. Effects like electrostatic attraction of anions by polycations (e.g. proteins) can prevent the equal distribution of a substance in water. Of course the same would be true for polyanions (nucleic acids) and cations. The resulting thermodynamic imbalance is then compensated by shifts in partial molar volume and therefore local density of water. This behaviour is unique for water and difficult to address experimentally since it is not measurable by classical physical methods.
In this theory low density water is supposed to have strong long range hydrogen bonds. They are weaker and more short-ranged in high density water. As a consequence the hydrophobic inner regions of a protein are better hydrated in high density water, denaturation is easier in the presence of weaker hydrogen bonds. If we now consider a solution of molecules which aggregate relatively large amounts of water in their hydration shell – like compatible solutes do – then this will create high density water fractions around the molecules and, subsequently, low density fractions elsewhere. Proteins in such an environment are now surrounded by low density water with strong, long range hydrogen bonds. This makes hydration of hydrophobic regions more difficult. Therefore, proteins are stabilized against unfolding.
The major drawback of this model is that the original idea behind it is based on structures of solid state water (ice) and experiments were done with gels. Therefore, we have to be very careful when trying to draw conclusions for the liquid state or even the situation in vivo. Nevertheless, though the model itself was not developed further, the concept is well worth considering [139, 140] especially when dealing with surface effects .
Hydrogen bonds, van der Waals, electrostatic and hydrophobic interactions were long known to be responsible for protein folding when Liu and Bolen  discovered an additional force. The osmophobic effect becomes relevant in highly concentrated solutions – and in organisms which require high intraellular concentrations of osmolytes. In contrast to the hydrophobic effect which causes apolar amino acid residues to aggregate in the protein interior the osmophobic effect influences the conformation of the peptide backbone. The peptide backbone is preferentially excluded from a compatible solute solution, thus stabilizing the conformation of the backbone and preventing protein denaturation. This is in accordance with the preferential interaction theory  where the solutes are excluded from the protein surface. But in addition to the overall effect of destabilization/stabilization, with this model we are able to explore single thermodynamic effects. The molar transfer free energy of a substance ΔGTR from water into a solute solution can be easily calculated as ΔGTR = R × T × ln(cW/cS), with gas constant R, absolute temperature T and the ratio of maximum concentrations of the substance in water cW and in solute solution cS.
Using individual amino acids and diketopiperazine (cyclic di-glycine) as peptide backbone model, Bolen and co-workers observed that the major player was the osmophobic effect (on the backbone) with hydrophobic (side chain) for fine tuning. A current publication of the Bolen group discusses the prediction of energetics in folding and unfolding .
Applying the protein models to nucleic acids
Current research on compatible solute protein interaction seems to converge from different points of view to a common concept. Both the Bolen and Record group postulate that the effects of osmolytes are based on the exclusion from or accumulation at polar peptide groups [142, 143], with Street  breaking down the interactions into more simple physical properties and Courtenay  being the first to quantify those effects. Exclusion and accumulation are related to accessibility of the targets of those interactions. The solvent (and therefore solute) accessible surface area plays an important role. This brings us back to the point, that not only preferential interaction and osmophobic effect but also water replacement and maybe even water density fractions  are all related to surface effects. But how does this help us to apply the protein/compatible solute models to nucleic acids? As we seem to have a common basis in the protein models, we need to investigate a commonality shared by nucleic acids and proteins.
As a first approach, we could assume that surface features and the accessibility of those features is similar for proteins and nucleic acids. Simplifying things even more, we could treat nucleic acids or nucleic acid/protein complexes as structures with a uniform surface, as assumed for proteins in the preferential interaction model. Of course neglect of surface structure details is a weak point of this model and controversially discussed for proteins. Experimental data show that glycine betaine and urea, which are both denaturants of double stranded DNA, behave differently with respect to interaction [145, 146]. While glycine betaine is preferredly excluded from the the negatively charged phosphate backbone , urea has to be regarded as an inert solute in a sense that its concentrations in the bulk and near the surface are the same. Different alcohols have been reported to be preferredly excluded from spermidine/DNA assemblies without affecting physical properties . Especially with nonpolar alcohols we observe exclusion based on repulsive hydration interactions with the charged DNA surface and depending on the balance between alkyl carbons and hydroxyl oxygens .
Further experimental data can be collected from those special situations of low water activity where the water replacement model might be applied, in particular from freeze drying of DNA. Lyophilized cationic lipid-DNA complexes are reported to be stabilized best by disaccharides, while polysaccharides had no effect [33, 150]. In a similar study with DNA loaded nanoparticles trehalose and glycerol were found to have the best effects among other sugars and polyols . Stabilization was contributed to water replacement by the solutes. In contrast to the above, naked DNA was damaged during lyophylization, even in the presence of sugars. However, when a polycation was added, protection from degradation after spray freeze drying  was observed. Apparently, water replacement plays a role in DNA stability at low water activity but there must be other factors behind the observed protective effects.
Obviously both models have their use, but as we see, they also have their limitations. This is also true when we turn to water density fractions. Although three of the nucleic acids bases (adenine, thymine and guanine) have relatively low solubility in water they are not really hydrophobic, we know that all bases form hydrogen bonds. Therefore, we would expect dissociation of double strands and similar structures to be easier in low density water. And indeed, as discussed above, a range of solutes which stabilize proteins against unfolding lower the melting point of DNA (e.g. [30, 43, 53, 73, 74]). But the model cannot be the sole explanation for osmolyte effects on nucleic acid/protein-complexes. Especially stabilization of such complexes  has to include additional aspects because solutes which stabilize proteins destabilize the nucleic acid, at least they do destabilize double helical structures in the sense that they lower TM. As demonstrated, lowering of the water activity by solutes might inhibit dissociation and override the destabilization [145, 146].
This leaves us with the osmophobic effect. Here the interesting question is, whether we have osmophobic effects at all with nucleic acids. Due to its extremely high solubility in water one would not suspect the highly polar phosphate/sugar polyanion to qualifiy as an osmophobic element. But the term osmophobic as used in the model rather relates to the behaviour of the (macro)molecule in the presence of cosolvents than to general solubility. In continuation of their research on ion/RNA interactions  the Draper group very recently investigated the influence of compatible solutes on a diversity of RNA structures . Using the concepts of Bolen and Record thermodynamics could be resolved sufficiently. Stabilization of RNA tertiary structure by glycine betaine was shown to correspond with complete exclusion of betaine from the backbone, which is exactly the same situation as for proteins. With such an effect we can also easily explain destabilization of dsDNA (resulting in a lower TM) if we postulate that corresponding ssDNA structures are more stable than the duplex in presence of a solute. But is this the whole story? It is admitted in the survey by Lambert that the model is "simplifying" the situation, especially with respect to counterions. And although all fits nicely for betaine we have already encountered dsDNA denaturants like urea that behave differently with respect to interaction with the backbone. As already shown more than 30 years ago [153, 154] in the presence of certain compounds nucleic acid bases tend to be more easily exposed in an environment, and we can draw the conclusion that protein stabilizing solutes which destabilize DNA may have a similar effect. So in addition to a possible osmophobic element, the nucleic acid backbone, we can characterize the bases as an osmophilicelement.
If we want to go beyond calculations and understand the mechanistics behind the complex interactions, even Drapers' concept, valuable as it is, leaves questions unanswered. Especially when it comes to the point of the counter ion atmosphere we are at the most striking difference between nucleic acids and proteins, which shows us that the surfaces of proteins and nucleic acids are in fact not very similar. We can draw on a wealth of information about ions in general, and also about the role of counterions or ionic strength in nucleic acid or nucleic acid/protein complex properties [25, 36, 50, 52, 59–62, 127]. The phosphate backbone or, to be more precise, its strong negative charge and the atmosphere connected to it, are very likely both interacting partners for (compatible) solutes. So we cannot leave this aspect unattended.
In this context we have to pay special attention to anioic organic solutes which are accumulated in particular by thermophilic and hyperthermophilic microrganisms , potassium presumably serving as counterion. Unfortunately we do know even less about the effects anionic solutes have on nucleic acids than we know about the effects of zwitterionic and uncharged species; nevertheless, positive effects in nucleic acid applications have been reported [4, 156].
Since nucleic acid structures are already destabilized by the elevated temperatures of (hyper-)thermophilic habitats an additional destabilizing effect would probably be fatal in vivo. The interesting question is, do anionic solutes have special properties to be able to stabilize both proteins and nucleic acids against thermal denaturation? Obviously, due to the negative charge of the phosphate backbone electrostatic repulsion makes direct interactions between nucleic acids and anionic solutes quite improbable. On the other hand, osmophilic or osmophobic effects do not necessarily depend on direct interactions but describe the general behaviour of a (macro)molecule in the presence of a (compatible) solute. And we should not forget about the potassium cations which might have to work in concert with the organic anion for the best effect in protein and DNA stabilization.
Things get even more exiting when looking at organisms dealing with thermal and osmotic stress since they have to balance adaptation to both conditions. We know that some of the organic anions like glycosylglycerate do have osmoprotective properties while others like mannosylglycerate are solely for thermoprotection  with trehalose serving as the main osmoprotectant . But a lack of experimental data does not allow for more than speculation.
A third point is that all the models presented above deal with possible indirect effects of CS on biopolymers, but at least tetrahydropyrimidines have also been reported to interact more directly with DNA [122, 123]. Interestingly, these compounds seem to prefer interactions with guanine, a property which perfectly correlates with their increased impact on GC-rich sequences. Thus, indirect interactions are not the whole story either. At a first glance, the structural similarity of the tetrahydropyrimidines with the pyrimidine bases may indicate the ability to intercalate into the base stacks. One should however be aware of the fact that these solutes are not completely planar but rather resemble a half-chair conformation. Trying to understand potential direct interactions of tetrahydropyrimidines with other biomolecules we can obtain useful information from protein biochemistry. The recent crystallization and X-ray analysis of substrate binding proteins of osmolyte transporters revealed a pocket of aromatic residues as the binding motif. In ProX  this pocket is formed by four tyrosines and in OpuAC by three tryptophanes . The fact that binding of glycine betaine and proline betaine is apparently established by cation-π-interactions and non-classical hydrogen bonds provides us with a means to anticipate direct interactions with the nucleic acid bases.
In addition we cannot fail to notice an influence of overall bulk dielectric constant ε' on DNA properties . And in this context it is known for a long time how drastic ε' can be changed by small organic compounds [160, 161].
So far there are neither publications proposing a general concept for thermodynamic calculations of compatible solute effects on nucleic acids nor molecular dynamics simulations thereof. A recent publication by Rösgen  discusses the basics of solute protein and solute protein metabolite thermodynamics and might prove to be a good starting point for similar considerations towards solute nucleic acid interactions. Closest to calculations concerning nucleic acids and solutes come a thermodynamic model including the effects of salt concentration on nucleic acid duplex-simplex transitions  and molecular dynamics studies of ion distributions for DNA duplexes and clusters . But as discussed above, the influence of solutes also arises from indirect effects, mainly as a consequence of a modulation of the properties of the solvent water. The thermodynamics of force-induced melting of DNA double helices, for example, including indirect effects of solvents and solutes [165, 166] are well known. In addition the thermodynamics of tRNA microhairpin stability and its solvent dependence have been investigated [90, 167]. Some earlier publications already discussed solvent effects on nucleic acid base associations and simulations thereof [168, 169] as well as general DNA dynamics in aqueous solutions . Modes of direct interaction of compatible solutes which display structural similarities to DNA, like the tetrahydropyrimidines, might be derived from considerations on DNA aggregates and their melting behaviour . More general models for DNA melting, which consider base pairing and stacking , bubble nucleation and cooperativity [86, 88] or bubble relaxation  and inhomogeneity of DNA melting  are permanently under improvement. And a closer investigation into the secondary structure of nucleic acids  and their involvement in nucleic acid/protein interactions  might help us to understand how compatible solutes affect nucleic acid/protein complexes and their formation.
Compatible solute effects on nucleic acid properties and nucleic acid/protein complexes have been known for some time now. Besides their exploitation for in vitro applications they are also of great significance in vivo and have a large impact on vital functions. In contrast to the large amount of application data, we know only little about the molecular background of the observed effects. What is missing is a broad systematic approach to basic data, especially such simple things like thermal melting and stability. Nevertheless, we do know more than one might suspect from a quick glance at present day research.
One major problem is the comparability of data, which even starts at simple thermal melting experiments. In a system where buffer composition strongly affects the properties of the subject under investigation we have to expect three- (or more-) way interactions between target (nucleic acid), buffer and effector (compatible solute). Therefore, a broader coherent set of basic data is definitely needed before we can start to interpret the results of more sophisticated experiments in a proper way, which is especially true for anionic organic solutes.
a) direct interactions with nucleic acid bases, probably due to cation-π-interactions similar to the situation in the solute binding proteins
b) direct interactions with the negatively charged phosphate backbone
c) interactions with the positively charged counterions and
d) indirect interactions via changes in solvent properties.
When investigating interactions with nucleic acid/protein-structures things get even more complex, since we have to consider not only the effects on nucleic acids, but also interactions of solutes with the protein(s) and the influence of osmolytes on the interactions between nucleic acids and proteins. In vivo of course, all other metabolites add to the whole picture.
We can use the concept that the effects of osmolytes are based on the exclusion or accumulation at surface elements for calculation of thermodynamics as demonstrated by Lambert , and all four of the models presented here have their – however limited – use for compatible solute/nucleic acid interactions. But – since those models are developed for protein/solute interactions – direct interactions are to a large extent excluded. Another point left out is the negative charge of the phosphate backbone and its counterion atmosphere. And lastly, in analogy to the osmophobic effect of proteins we might want to mint a new term here: The potential osmophilic effect of nucleic acid bases.
Many thanks go to Erwin Galinski and Ute Habermann, University Bonn, Germany, for fruitful discussions and for critically reading the manuscript. Additional thanks go to Peter Voss, University Duisburg, Germany, for introducing me to the protein/solute-interaction models.
- Galinski EA: Osmoadaptation in bacteria. Adv Microb Physiol. 1995, 37: 272-328.Google Scholar
- da Costa MS, Santos H, Galinski EA: An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv Biochem Eng Biotechnol. 1998, 61: 117-153.Google Scholar
- Roberts MF: Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Systems. 2005, 1: 5-10.1186/1746-1448-1-5.Google Scholar
- Lentzen G, Schwarz T: Extremolytes: Natural compounds from extremophiles for versatile applications. Appl Microbiol Biotechnol. 2006, 72: 623-634. 10.1007/s00253-006-0553-9.Google Scholar
- Galinski EA, Pfeiffer HP, Trüper HG: 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem. 1985, 149: 135-139. 10.1111/j.1432-1033.1985.tb08903.x.Google Scholar
- Ventosa A, Nieto JJ, Oren A: Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev. 1998, 62: 504-544.Google Scholar
- Petrovic U, Gunde-Cimerman N, Plemenitas A: Cellular responses to environmental salinity in the halophilic black yeast Hortaea werneckii. Mol Microbiol. 2002, 45: 665-672. 10.1046/j.1365-2958.2002.03021.x.Google Scholar
- Oren A: Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev. 1999, 63: 334-348.Google Scholar
- Patzelt H: Hydrocarbon Degradation under Hypersaline Conditions. Adaptation to life at high salt concentrations in archaea, bacteria, and eukarya. Edited by: Gunde-Cimerman N, Oren A, Plemenitas A. 2005, SpringerGoogle Scholar
- Lindemose S, Nielsen PE, Mollegaard NE: Polyamines preferentially interact with bent adenine tracts in double-stranded DNA. Nucleic Acids Res. 2005, 33: 1790-1803. 10.1093/nar/gki319.Google Scholar
- Cornacchione S, Sadick NS, Neveu M, Talbourdet S, Lazou K, Viron C, Renimel I, de Quéral D, Kurfurst R, Schnebert S, Heusèle C, André P, Perrier E: In vivo skin antioxidant effect of a new combination based on a specific Vitis vinifera shoot extract and a biotechnological extract. J Drugs Dermatol. 2007, 6: s8-13.Google Scholar
- Heinrich U, Garbe B, Tronnier H: In vivo assessment of Ectoin: a randomized, vehicle-controlled clinical trial. Skin Pharmacol Physiol. 2007, 20: 211-218. 10.1159/000103204.Google Scholar
- Klein J, Schwarz T, Lentzen G: Ectoine as a natural component of food: detection in red smear cheeses. J Dairy Res. 2007, 1-6.Google Scholar
- Lippert K, Galinski EA: Enzyme stabilization by ectoine-type compatible solutes: Protection against heating, freezing and drying. Appl Microbiol Biotech. 1992, 37: 61-65. 10.1007/BF00174204.Google Scholar
- Knapp S, Ladenstein R, Galinski EA: Extrinsic protein stabilization by the naturally occurring osmolytes beta-hydroxyectoine and betaine. Extremophiles. 1999, 3: 191-198. 10.1007/s007920050116.Google Scholar
- Göller K, Galinski EA: Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute addition. J Mol Catal B: Enzymatic. 1999, 7: 37-54. 10.1016/S1381-1177(99)00043-0.Google Scholar
- Andersson MM, Breccia JD, Hatti-Kaul R: Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase. Biotechnol Appl Biochem. 2000, 32 (Pt 3): 145-153. 10.1042/BA20000014.Google Scholar
- Barth S, Huhn M, Matthey B, Klimka A, Galinski EA, Engert A: Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Appl Environ Microbiol. 2000, 66: 1572-1579. 10.1128/AEM.66.4.1572-1579.2000.Google Scholar
- Louis P, Trüper HG, Galinski EA: Survival of Escherichia coli during drying and storage in the presence of compatible solu.tes. Appl Microbiol Biotechnol. 1994, 41: 684-688. 10.1007/BF00167285.Google Scholar
- Hoeckstra FA, Wolkers WF, Buitink J, Golowina EA, Crowe JH, Crowe LM: Membrane stabilisation in the dry state. Comp Biochem Physiol. 1997, 116A: 335-341. 10.1016/S0300-9629(96)00272-1.Google Scholar
- Arakawa T, Timasheff SN: Preferential interactions of proteins with solvent components in aqueous amino acid solutions. Arch Biochem Biophys. 1983, 224: 169-177. 10.1016/0003-9861(83)90201-1.Google Scholar
- Clegg JS, Seitz P, Seitz W, Hazlewood CF: Cellular responses to extreme water loss: the water-replacement hypothesis. Cryobiology. 1982, 19: 306-316. 10.1016/0011-2240(82)90159-6.Google Scholar
- Wiggins PM: Role of water in some biological processes. Microbiol Rev. 1990, 54: 432-449.Google Scholar
- Liu Y, Bolen DW: The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes. Biochemistry. 1995, 34: 12884-12891. 10.1021/bi00039a051.Google Scholar
- Lambert D, Draper DE: Effects of osmolytes on RNA secondary and tertiary structure stabilities and RNA-Mg2+ interactions. J Mol Biol. 2007, 370: 993-1005. 10.1016/j.jmb.2007.03.080.Google Scholar
- Baskaran N, Kandpal RP, Bhargava AK, Glynn MW, Bale A, Weissman SM: Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 1996, 6: 633-638. 10.1101/gr.6.7.633.Google Scholar
- Weissensteiner T, Lanchbury JS: Strategy for controlling preferential amplification and avoiding false negatives in PCR typing. Biotechniques. 1996, 21: 1102-1108.Google Scholar
- Henke W, Loening SA: Recently, betaine has been introduced as an additive in different PCR strategies. Nucleic Acids Res. 1998, 26: 687-Google Scholar
- Shammas FV, Heikkila R, Osland A: Improvement of quantitative PCR reproducibility by betaine as determined by fluorescence-based method. Biotechniques. 2001, 30: 950-954.Google Scholar
- Spiess A, Mueller N, Ivell R: Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin Chem. 2004, 50: 1256-1259. 10.1373/clinchem.2004.031336.Google Scholar
- Schnoor M, Voss P, Cullen P, Boking T, Galla H, Galinski EA, Lorkowski S: Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer. Biochem Biophys Res Commun. 2004, 322: 867-872. 10.1016/j.bbrc.2004.07.200.Google Scholar
- Sahdev S, Saini S, Tiwari P, Saxena S, Singh Saini K: Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. Mol Cell Probes. 2007, 21: 303-307. 10.1016/j.mcp.2007.03.004.Google Scholar
- Allison SD, Anchordoquy TJ: Mechanisms of protection of cationic lipid-DNA complexes during lyophilization. J Pharm Sci. 2000, 89: 682-691. 10.1002/(SICI)1520-6017(200005)89:5<682::AID-JPS14>3.0.CO;2-#.Google Scholar
- Semrad K, Green R: Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA. RNA. 2002, 8: 401-411. 10.1017/S1355838202029722.Google Scholar
- Potty ASR, Fu JY, Balan S, Haymore BL, Hill DJ, Fox GE, Willson RC: Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: role of water activity. J Chromatogr A. 2006, 1115: 88-92. 10.1016/j.chroma.2006.02.077.Google Scholar
- Hethke C, Bergerat A, Hausner W, Forterre P, Thomm M: Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription. Genetics. 1999, 152: 1325-1333.Google Scholar
- Brigotti M, Petronini PG, Carnicelli D, Alfieri RR, Bonelli MA, Borghetti AF, Wheeler KP: Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. Biochem J. 2003, 369: 369-374. 10.1042/BJ20021056.Google Scholar
- Malin G, Iakobashvili R, Lapidot A: Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system. J Biol Chem. 1999, 274: 6920-6929. 10.1074/jbc.274.11.6920.Google Scholar
- Sugimoto K, Makihara T, Saito A, Ohishi N, Nagase T, Takai D: Betaine improved restriction digestion. Biochem Biophys Res Commun. 2005, 337: 1027-1029. 10.1016/j.bbrc.2005.09.145.Google Scholar
- Bachrach U: Naturally occurring polyamines: interaction with macromolecules. Curr Protein Pept Sci. 2005, 6: 559-566. 10.2174/138920305774933240.Google Scholar
- Flock S, Labarbe R, Houssier C: Osmotic effectors and DNA structure: effect of glycine on precipitation of DNA by multivalent cations. J Biomol Struct Dyn. 1995, 13: 87-102.Google Scholar
- Bhattacharya S, Mandal SS: Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting. Biochim Biophys Acta. 1997, 1323: 29-44. 10.1016/S0005-2736(96)00171-X.Google Scholar
- Chandramouli KH, Thimmaiah KN, Chandrashekar A, D'Souza CJM: Interaction of 2-chloro-N10-substituted phenoxazine with DNA and effect on DNA melting. Nucleosides Nucleotides Nucleic Acids. 2004, 23: 1639-1656. 10.1081/NCN-200031461.Google Scholar
- Rich A: DNA comes in many forms. Gene. 1993, 135: 99-109. 10.1016/0378-1119(93)90054-7.Google Scholar
- Felsenfeld G, Rich A: Studies on the formation of two- and three-stranded polyribonucleotides. Biochim Biophys Acta. 1957, 26: 457-468. 10.1016/0006-3002(57)90091-4.Google Scholar
- Watson JD, Crick FH: Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 1953, 171: 737-738. 10.1038/171737a0.Google Scholar
- Barrick JE, Corbino KA, Winkler WC, Nahvi A, Mandal M, Collins J, Lee M, Roth A, Sudarsan N, Jona I, Wickiser JK, Breaker RR: New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc Natl Acad Sci USA. 2004, 101: 6421-6426. 10.1073/pnas.0308014101.Google Scholar
- Schwalbe H, Buck J, Furtig B, Noeske J, Wohnert J: Structures of RNA switches: insight into molecular recognition and tertiary structure. Angew Chem Int Ed Engl. 2007, 46: 1212-1219. 10.1002/anie.200604163.Google Scholar
- Mandal M, Lee M, Barrick JE, Weinberg Z, Emilsson GM, Ruzzo WL, Breaker RR: A glycine-dependent riboswitch that uses cooperative binding to control gene expression. Science. 2004, 306: 275-279. 10.1126/science.1100829.Google Scholar
- Draper DE, Grilley D, Soto AM: Ions and RNA folding. Annu Rev Biophys Biomol Struct. 2005, 34: 221-243. 10.1146/annurev.biophys.34.040204.144511.Google Scholar
- Frank-Kamenetskii F: Simplification of the empirical relationship between melting temperature of DNA, its GC content and concentration of sodium ions in solution. Biopolymers. 1971, 10: 2623-2624. 10.1002/bip.360101223.Google Scholar
- Volker J, Klump HH, Manning GS, Breslauer KJ: Counterion association with native and denatured nucleic acids: an experimental approach. J Mol Biol. 2001, 310: 1011-1025. 10.1006/jmbi.2001.4841.Google Scholar
- Rees WA, Yager TD, Korte J, von Hippel PH: Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 1993, 32: 137-144. 10.1021/bi00052a019.Google Scholar
- Flock S, Labarbe R, Houssier C: 23Na NMR study of the effect of organic osmolytes on DNA counterion atmosphere. Biophys J. 1996, 71: 1519-1529.Google Scholar
- Houssier C, Gilles R, Flock S: Effects of compensatory solutes on DNA and chromatin structural organization in solution. Comp Biochem Physiol A Physiol. 1997, 117: 313-318. 10.1016/S0300-9629(96)00269-1.Google Scholar
- Manning GS: Limiting Laws and Counterion Condensation in Polyelectrolyte Solutions I. Colligative Properties. J Chem Phys. 1969, 51: 127-1250.Google Scholar
- Oosawa F: Polyelectrolytes. 1971, Marcel Dekker, New YorkGoogle Scholar
- Shaughnessy B, Yang Q: Manning-Oosawa Counterion Condensation. Phys Rev Letters. 2005, 94: 0483021-0483024. 10.1103/PhysRevLett.94.048302.Google Scholar
- Hackl EV, Blagoi YP: The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions. Biopolymers. 2005, 77: 315-324. 10.1002/bip.20225.Google Scholar
- Sundaresan N, Thomas T, Thomas TJ, Pillai CKS: Lithium ion induced stabilization of the liquid crystalline DNA. Macromol Biosci. 2006, 6: 27-32. 10.1002/mabi.200500145.Google Scholar
- Miyoshi D, Nakao A, Sugimoto N: Structural transition from antiparallel to parallel G-quadruplex of d(G4T4G4) induced by Ca2+. Nucleic Acids Res. 2003, 31: 1156-1163. 10.1093/nar/gkg211.Google Scholar
- Lavelle L, Fresco JR: Stabilization of nucleic acid triplexes by high concentrations of sodium and ammonium salts follows the Hofmeister series. Biophys Chem. 2003, 105: 681-699. 10.1016/S0301-4622(03)00097-8.Google Scholar
- Bergqvist S, O'Brien R, Ladbury JE: Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon. Biochemistry. 2001, 40: 2419-2425. 10.1021/bi002488m.Google Scholar
- Bergqvist S, Williams MA, O'Brien R, Ladbury JE: Reversal of halophilicity in a protein-DNA interaction by limited mutation strategy. Structure. 2002, 10: 629-637. 10.1016/S0969-2126(02)00749-9.Google Scholar
- Bergqvist S, Williams MA, O'Brien R, Ladbury JE: Halophilic adaptation of protein-DNA interactions. Biochem Soc Trans. 2003, 31: 677-680. 10.1042/BST0310677.Google Scholar
- Oren A, Heldal M, Norland S, Galinski EA: Intracellular ion and organic solute concentrations of the extremely halophilic bacterium Salinibacter ruber. Extremophiles. 2002, 6: 491-498. 10.1007/s00792-002-0286-3.Google Scholar
- Kraegeloh A, Kunte HJ: Novel insights into the role of potassium for osmoregulation in Halomonas elongata. Extremophiles. 2002, 6: 453-462. 10.1007/s00792-002-0277-4.Google Scholar
- Kraegeloh A, Amendt B, Kunte HJ: Potassium transport in a halophilic member of the bacteria domain: identification and characterization of the K+ uptake systems TrkH and TrkI from Halomonas elongata DSM 2581T. J Bacteriol. 2005, 187: 1036-1043. 10.1128/JB.187.3.1036-1043.2005.Google Scholar
- Wallace RB, Shaffer J, Murphy RF, Bonner J, Hirose T, Itakura K: Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 1979, 6: 3543-3557. 10.1093/nar/6.11.3543.Google Scholar
- Korolev N, Lyubartsev AP, Nordenskiold L: Application of the Poisson Boltzmann polyelectrolyte model for analysis of thermal denaturation of DNA in the presence of Na+ and polyamine cations. Biophys Chem. 2003, 104: 55-66. 10.1016/S0301-4622(02)00338-1.Google Scholar
- Leber M, Kaderali L, Schonhuth A, Schrader R: A fractional programming approach to efficient DNA melting temperature calculation. Bioinformatics. 2005, 21: 2375-2382. 10.1093/bioinformatics/bti379.Google Scholar
- Everaers R, Kumar S, Simm C: Unified description of poly- and oligonucleotide DNA melting: nearest-neighbor, Poland-Sheraga, and lattice models. Phys Rev E Stat Nonlin Soft Matter Phys. 2007, 75: 041918-Google Scholar
- Rajendrakumar CS, Suryanarayana T, Reddy AR: DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process. FEBS Lett. 1997, 410: 201-205. 10.1016/S0014-5793(97)00588-7.Google Scholar
- Lavelle L, Fresco JR: Enhanced stabilization of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21) and poly r(U:AU) by water structure-making solutes. Biophys Chem. 2003, 105: 701-720. 10.1016/S0301-4622(03)00098-X.Google Scholar
- Kumar N, Maiti S: The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study. Nucleic Acids Res. 2005, 33: 6723-6732. 10.1093/nar/gki961.Google Scholar
- Smirnov IV, Shafer RH: Electrostatics dominate quadruplex stability. Biopolymers. 2007, 85: 91-101. 10.1002/bip.20609.Google Scholar
- Miyoshi D, Nakao A, Sugimoto N: Molecular crowding regulates the structural switch of the DNA G-quadruplex. Biochemistry. 2002, 41: 15017-15024. 10.1021/bi020412f.Google Scholar
- Miyoshi D, Nakao A, Sugimoto N: Effect of putrescine and PEG on a structural transition of DNA G-quadruplex. Nucleosides Nucleotides Nucleic Acids. 2003, 22: 1591-1594. 10.1081/NCN-120023041.Google Scholar
- Anchordoquy TJ, Armstrong TK, Molina MDC: Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes. J Pharm Sci. 2005, 94: 1226-1236. 10.1002/jps.20353.Google Scholar
- Lazurkin YS, Frank-Kamenetskii MD, Trifonov EN: Melting of DNA: its study and application as a research method. Biopolymers. 1970, 9: 1253-1306. 10.1002/bip.1970.360091102.Google Scholar
- Pohorille A, Ross WS, Tinoco IJ: DNA dynamics in aqueous solution: opening the double helix. Int J Supercomput Appl. 1990, 4: 81-96.Google Scholar
- Mergny J, Lacroix L: Analysis of thermal melting curves. Oligonucleotides. 2003, 13: 515-537. 10.1089/154545703322860825.Google Scholar
- Li W, Xi B, Yang W, Hawkins M, Schubart UK: Complex DNA melting profiles of small PCR products revealed using SYBR Green I. Biotechniques. 2003, 35: 702-4. 706Google Scholar
- Ivanov V, Zeng Y, Zocchi G: Statistical mechanics of base stacking and pairing in DNA melting. Phys Rev E Stat Nonlin Soft Matter Phys. 2004, 70: 051907-Google Scholar
- Yakovchuk P, Protozanova E, Frank-Kamenetskii MD: Base-stacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Res. 2006, 34: 564-574. 10.1093/nar/gkj454.Google Scholar
- Ares S, Voulgarakis NK, Rasmussen KO, Bishop AR: Bubble nucleation and cooperativity in DNA melting. Phys Rev Lett. 2005, 94: 035504-10.1103/PhysRevLett.94.035504.Google Scholar
- Bicout DJ, Kats E: Bubble relaxation dynamics in double-stranded DNA. Phys Rev E Stat Nonlin Soft Matter Phys. 2004, 70: 010902-Google Scholar
- Ivanov V, Piontkovski D, Zocchi G: Local cooperativity mechanism in the DNA melting transition. Phys Rev E Stat Nonlin Soft Matter Phys. 2005, 71: 041909-Google Scholar
- Spink CH, Chaires JB: Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA. Biochemistry. 1999, 38: 496-508. 10.1021/bi9820154.Google Scholar
- Biala E, Strazewski P: Internally mismatched RNA: pH and solvent dependence of the thermal unfolding of tRNA(Ala) acceptor stem microhairpins. J Am Chem Soc. 2002, 124: 3540-3545. 10.1021/ja0161305.Google Scholar
- Hammouda B, Worcester D: The Denaturation Transition of DNA in Mixed Solvents. Biophys J. 2006Google Scholar
- Bjorndal MT, Fygenson DK: DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay. Biopolymers. 2002, 65: 40-44. 10.1002/bip.10220.Google Scholar
- Giglio S, Monis PT, Saint CP: Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res. 2003, 31: e136-10.1093/nar/gng135.Google Scholar
- Spink CH, Garbett N, Chaires JB: Enthalpies of DNA melting in the presence of osmolytes. Biophys Chem. 2007, 126: 176-185. 10.1016/j.bpc.2006.07.013.Google Scholar
- Abu Al-Soud W, Radstrom P: Capacity of nine thermostable DNA polymerases To mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol. 1998, 64: 3748-3753.Google Scholar
- Ignatov KB, Miroshnikov AI, Kramarov VM: [A new approach to enhance PCR specificity]. Bioorg Khim. 2003, 29: 403-407.Google Scholar
- Hengen PN: Optimizing multiplex and LA-PCR with betaine. Trends Biochem Sci. 1997, 22: 225-226. 10.1016/S0968-0004(97)01069-4.Google Scholar
- Shammas FV, Heikkila R, Osland A: Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR. Clin Chim Acta. 2001, 304: 19-28. 10.1016/S0009-8981(00)00374-0.Google Scholar
- Kang J, Lee MS, Gorenstein DG: The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods. 2005, 64: 147-151. 10.1016/j.jbbm.2005.06.003.Google Scholar
- Ralser M, Querfurth R, Warnatz H, Lehrach H, Yaspo M, Krobitsch S: An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 2006, 347: 747-751. 10.1016/j.bbrc.2006.06.151.Google Scholar
- Shi X, Jarvis DL: A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes. Anal Biochem. 2006, 356: 222-228. 10.1016/j.ab.2006.06.028.Google Scholar
- Abu Al-Soud W, Radstrom P: Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol. 2000, 38: 4463-4470.Google Scholar
- Diakou A, Dovas CI: Optimization of random-amplified polymorphic DNA producing amplicons up to 8500 bp and revealing intraspecies polymorphism in Leishmania infantum isolates. Anal Biochem. 2001, 288: 195-200. 10.1006/abio.2000.4896.Google Scholar
- Chen X, Zhang X, Liang R, Cao M: Betaine improves LA-PCR amplification. Sheng Wu Gong Cheng Xue Bao. 2004, 20: 715-718.Google Scholar
- Iakobashvili R, Lapidot A: Low temperature cycled PCR protocol for Klenow fragment of DNA polymerase I in the presence of proline. Nucleic Acids Res. 1999, 27: 1566-1568. 10.1093/nar/27.6.1566.Google Scholar
- Hirano M, Nakamura S, Mitsunaga F, Okada M, Shirahama S, Eberle R: One-step PCR to distinguish B virus from related primate alphaherpesviruses. Clin Diagn Lab Immunol. 2002, 9: 716-719. 10.1128/CDLI.9.3.716-719.2002.Google Scholar
- Carninci P, Nishiyama Y, Westover A, Itoh M, Nagaoka S, Sasaki N, Okazaki Y, Muramatsu M, Hayashizaki Y: Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc Natl Acad Sci USA. 1998, 95: 520-524. 10.1073/pnas.95.2.520.Google Scholar
- Louwrier A, Valk van der A: Can sucrose affect polymerase chain reaction product formation?. Biotechnology Letters. 2001, 23: 175-178. 10.1023/A:1005656100993.Google Scholar
- Spiess A, Ivell R: A highly efficient method for long-chain cDNA synthesis using trehalose and betaine. Anal Biochem. 2002, 301: 168-174. 10.1006/abio.2001.5474.Google Scholar
- Chakrabarti R, Schutt CE: The enhancement of PCR amplification by low molecular-weight sulfones. Gene. 2001, 274: 293-298. 10.1016/S0378-1119(01)00621-7.Google Scholar
- Chakrabarti R, Schutt CE: The enhancement of PCR amplification by low molecular weight amides. Nucleic Acids Res. 2001, 29: 2377-2381. 10.1093/nar/29.11.2377.Google Scholar
- Chakrabarti R, Schutt CE: Novel sulfoxides facilitate GC-rich template amplification. Biotechniques. 2002, 32: 866, 868, 870–874Google Scholar
- Thakar M, Bilenko A, Becktel WJ: Osmolyte mediation of T7 DNA polymerase and plasmid DNA stability. Biochemistry. 1994, 33: 12255-12259. 10.1021/bi00206a031.Google Scholar
- McDowell DG, Burns NA, Parkes HC: Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR. Nucleic Acids Res. 1998, 26: 3340-3347. 10.1093/nar/26.14.3340.Google Scholar
- Mytelka DS, Chamberlin MJ: Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus. Nucleic Acids Res. 1996, 24: 2774-2781. 10.1093/nar/24.14.2774.Google Scholar
- Bachmann HS, Siffert W, Frey UH: Successful amplification of extremely GC-rich promoter regions using a novel 'slowdown PCR' technique. Pharmacogenetics. 2003, 13: 759-766. 10.1097/00008571-200312000-00006.Google Scholar
- Record MTJ, Courtenay ES, Cayley S, Guttman HJ: Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments. Trends Biochem Sci. 1998, 23: 190-194. 10.1016/S0968-0004(98)01207-9.Google Scholar
- Pul U, Wurm R, Wagner R: The role of LRP and H-NS in transcription regulation: involvement of synergism, allostery and macromolecular crowding. J Mol Biol. 2007, 366: 900-915. 10.1016/j.jmb.2006.11.067.Google Scholar
- Sidorova NY, Muradymov S, Rau DC: Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays. Nucleic Acids Res. 2005, 33: 5145-5155. 10.1093/nar/gki808.Google Scholar
- Sidorova NY, Rau DC: Removing water from an EcoRI-noncognate DNA complex with osmotic stress. J Biomol Struct Dyn. 1999, 17: 19-31.Google Scholar
- Sidorova NY, Rau DC: The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity. Biopolymers. 2000, 53: 363-368. 10.1002/(SICI)1097-0282(20000415)53:5<363::AID-BIP1>3.0.CO;2-Q.Google Scholar
- Inbar L, Frolow F, Lapidot A: The conformation of new tetrahydropyrimidine derivatives in solution and in the crystal. Eur J Biochem. 1993, 214: 897-906. 10.1111/j.1432-1033.1993.tb17993.x.Google Scholar
- Lapidot A, Inbar L, Aloni Y, Ben-Asher E: Purified tetrahydropyrimidine derivatives and pharmaceutical compositions comprising them. european patent application. 1993, EP 0553884A1.Google Scholar
- Lapidot A, Ben-Asher E, Eisenstein M: Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine-containing peptide, to HIV TAR RNA in vitro. FEBS Lett. 1995, 367: 33-38. 10.1016/0014-5793(95)00514-A.Google Scholar
- Lee SJ, Gralla JD: Osmo-regulation of bacterial transcription via poised RNA polymerase. Mol Cell. 2004, 14: 153-162. 10.1016/S1097-2765(04)00202-3.Google Scholar
- Gralla JD, Vargas DR: Potassium glutamate as a transcriptional inhibitor during bacterial osmoregulation. EMBO J. 2006, 25: 1515-1521. 10.1038/sj.emboj.7601041.Google Scholar
- Romeo Y, Bouvier J, Gutierrez C: Osmotic regulation of transcription in Lactococcus lactis: ionic strength-dependent binding of the BusR repressor to the busA promoter. FEBS Lett. 2007, 581: 3387-3390. 10.1016/j.febslet.2007.06.037.Google Scholar
- Fukue Y, Sumida N, Tanase J, Ohyama T: A highly distinctive mechanical property found in the majority of human promoters and its transcriptional relevance. Nucleic Acids Res. 2005, 33: 3821-3827. 10.1093/nar/gki700.Google Scholar
- de Souza FP, Neto AA, Fossey MA, Neto JR: The effect of changes in the bulk dielectric constant on the DNA torsional properties. Biopolymers. 2007, 87: 244-248. 10.1002/bip.20830.Google Scholar
- Hermann T, Patel DJ: Adaptive recognition by nucleic acid aptamers. Science. 2000, 287: 820-825. 10.1126/science.287.5454.820.Google Scholar
- Ulrich H, Trujillo CA, Nery AA, Alves JM, Majumder P, Resende RR, Martins AH: DNA and RNA aptamers: from tools for basic research towards therapeutic applications. Comb Chem High Throughput Screen. 2006, 9: 619-632. 10.2174/138620706778249695.Google Scholar
- Timasheff SN: In disperse solution, "osmotic stress" is a restricted case of preferential interactions. Proc Natl Acad Sci USA. 1998, 95: 7363-7367. 10.1073/pnas.95.13.7363.Google Scholar
- Timasheff SN: Protein hydration, thermodynamic binding, and preferential hydration. Biochemistry. 2002, 41: 13473-13482. 10.1021/bi020316e.Google Scholar
- Arakawa T, Tsumoto K, Nagase K, Ejima D: The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. Protein Expr Purif. 2007, 54: 110-116. 10.1016/j.pep.2007.02.010.Google Scholar
- Yu I, Jindo Y, Nagaoka M: Microscopic understanding of preferential exclusion of compatible solute ectoine: direct interaction and hydration alteration. J Phys Chem B. 2007, 111: 10231-10238. 10.1021/jp068367z.Google Scholar
- Kanias T, Acker J: Mammalian cell desiccation: Facing the challenges. Cell Pres Tech. 2006, 4: 253-277. 10.1089/cpt.2006.9996.Google Scholar
- Wiggins PM: High and low density water and resting, active and transformed cells. Cell Biol Int. 1996, 20: 429-435. 10.1006/cbir.1996.0054.Google Scholar
- Wiggins PM: High and low density intracellular water. Cell Mol Biol (Noisy-le-grand). 2001, 47: 735-744.Google Scholar
- Shepherd VA: The cytomatrix as a cooperative system of macromolecular and water networks. Curr Top Dev Biol. 2006, 75: 171-223. 10.1016/S0070-2153(06)75006-2.Google Scholar
- Zhao H, Olubajo O, Song Z, Sims AL, Person TE, Lawal RA, Holley LA: Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions. Bioorg Chem. 2006, 34: 15-25. 10.1016/j.bioorg.2005.10.004.Google Scholar
- Gun'ko VM, Turov VV, Bogatyrev VM, Zarko VI, Leboda R, Goncharuk EV, Novza AA, Turov AV, Chuiko AA: Unusual properties of water at hydrophilic/hydrophobic interfaces. Adv Colloid Interface Sci. 2005, 118: 125-172.Google Scholar
- Auton M, Bolen DW: Predicting the energetics of osmolyte-induced protein folding/unfolding. Proc Natl Acad Sci USA. 2005, 102: 15065-15068. 10.1073/pnas.0507053102.Google Scholar
- Street TO, Bolen DW, Rose GD: A molecular mechanism for osmolyte-induced protein stability. Proc Natl Acad Sci USA. 2006, 103: 13997-14002. 10.1073/pnas.0606236103.Google Scholar
- Courtenay ES, Capp MW, Record MTJ: Thermodynamics of interactions of urea and guanidinium salts with protein surface: relationship between solute effects on protein processes and changes in water-accessible surface area. Protein Sci. 2001, 10: 2485-2497. 10.1110/ps.ps.20801.Google Scholar
- Hong J, Capp MW, Anderson CF, Record MT: Preferential interactions in aqueous solutions of urea and KCl. Biophys Chem. 2003, 105: 517-532. 10.1016/S0301-4622(03)00112-1.Google Scholar
- Hong J, Capp MW, Anderson CF, Saecker RM, Felitsky DJ, Anderson MW, Record MTJ: Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability. Biochemistry. 2004, 43: 14744-14758. 10.1021/bi049096q.Google Scholar
- Felitsky DJ, Cannon JG, Capp MW, Hong J, Van Wynsberghe AW, Anderson CF, Record MTJ: The exclusion of glycine betaine from anionic biopolymer surface: why glycine betaine is an effective osmoprotectant but also a compatible solute. Biochemistry. 2004, 43: 14732-14743. 10.1021/bi049115w.Google Scholar
- Hultgren A, Rau DC: Exclusion of alcohols from spermidine-DNA assemblies: probing the physical basis of preferential hydration. Biochemistry. 2004, 43: 8272-8280. 10.1021/bi049559s.Google Scholar
- Stanley C, Rau DC: Preferential hydration of DNA: the magnitude and distance dependence of alcohol and polyol interactions. Biophys J. 2006, 91: 912-920. 10.1529/biophysj.106.086579.Google Scholar
- Li B, Li S, Tan Y, Stolz DB, Watkins SC, Block LH, Huang L: Lyophilization of cationic lipid-protamine-DNA (LPD) complexes. J Pharm Sci. 2000, 89: 355-364. 10.1002/(SICI)1520-6017(200003)89:3<355::AID-JPS7>3.0.CO;2-H.Google Scholar
- Sameti M, Bohr G, Ravi Kumar MNV, Kneuer C, Bakowsky U, Nacken M, Schmidt H, Lehr C: Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery. Int J Pharm. 2003, 266: 51-60. 10.1016/S0378-5173(03)00380-6.Google Scholar
- Kuo JS, Hwang R: Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA. J Pharm Pharmacol. 2004, 56: 27-33. 10.1211/0022357022494.Google Scholar
- Herskovits TT, Harrington JP: Solution studies of the nucleic acid bases and related model compounds. Solubility in aqueous alcohol and glycol solutions. Biochemistry. 1972, 11: 4800-4811. 10.1021/bi00775a025.Google Scholar
- Herskovits TT, Bowen JJ: Solution studies of the nucleic acid bases and related model compounds. Solubility in aqueous urea and amide solutions. Biochemistry. 1974, 13: 5474-5483. 10.1021/bi00724a004.Google Scholar
- Empadinhas N, da Costa MS: Diversity and biosynthesis of compatible solutes in hyper/thermophiles. Int Microbiol. 2006, 9: 199-206.Google Scholar
- Mascellani N, Liu X, Rossi S, Marchesini J, Valentini D, Arcelli D, Taccioli C, Helmer Citterich M, Liu C, Evangelisti R, Russo G, Santos JM, Croce CM, Volinia S: Compatible solutes from hyperthermophiles improve the quality of DNA microarrays. BMC Biotechnol. 2007, 7: 82-10.1186/1472-6750-7-82.Google Scholar
- Silva Z, Alarico S, Nobre A, Horlacher R, Marugg J, Boos W, Mingote AI, da Costa MS: Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose. J Bacteriol. 2003, 185: 5943-5952. 10.1128/JB.185.20.5943-5952.2003.Google Scholar
- Schiefner A, Holtmann G, Diederichs K, Welte W, Bremer E: Structural basis for the binding of compatible solutes by ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus. J Biol Chem. 2004, 279: 48270-48281. 10.1074/jbc.M403540200.Google Scholar
- Horn C, Sohn-Bosser L, Breed J, Welte W, Schmitt L, Bremer E: Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine. J Mol Biol. 2006, 357: 592-606. 10.1016/j.jmb.2005.12.085.Google Scholar
- Wyman J, McMeekin TL: The Dielectric Constants of Solutions of Amino Acids and Peptides. J Am Chem Soc. 1933, 55: 908-914. 10.1021/ja01330a006.Google Scholar
- Wyman J: The dielectric constant of solutions of dipolar ions. Chem Rev. 1936, 19: 213-239. 10.1021/cr60064a005.Google Scholar
- Rösgen J: Molecular basis of osmolyte effects on protein and metabolites. Methods Enzymol. 2007, 428: 459-486. 10.1016/S0076-6879(07)28026-7.Google Scholar
- Owczarzy R, Dunietz I, Behlke MA, Klotz IM, Walder JA: Thermodynamic treatment of oligonucleotide duplex-simplex equilibria. Proc Natl Acad Sci USA. 2003, 100: 14840-14845. 10.1073/pnas.2335948100.Google Scholar
- Long H, Kudlay A, Schatz GC: Molecular dynamics studies of ion distributions for DNA duplexes and DNA clusters: salt effects and connection to DNA melting. J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2006, 110: 2918-2926.Google Scholar
- Rouzina I, Bloomfield VA: Force-induced melting of the DNA double helix 1. Thermodynamic analysis. Biophys J. 2001, 80: 882-893.Google Scholar
- Rouzina I, Bloomfield VA: Force-induced melting of the DNA double helix. 2. Effect of solution conditions. Biophys J. 2001, 80: 894-900.Google Scholar
- Biala E, McClain W, Strazewski P: Thermodynamics of site-specific variant tRNA(Ala) acceptor stem microhairpins. Nucleosides Nucleotides. 1999, 18: 1575-1576. 10.1080/07328319908044788.Google Scholar
- Pohorille A, Pratt LR, Burt SK, MacElroy RD: Solution influence on biomolecular equilibria: nucleic acid base associations. J Biomol Struct Dyn. 1984, 1: 1257-1280.Google Scholar
- Pohorille A, Burt SK, MacElroy RD: Monte Carlo simulation of the influence of solvent on nucleic acid base associations. J Am Chem Soc. 1984, 106: 402-409. 10.1021/ja00314a025.Google Scholar
- Cherstvy AG, Kornyshev AA: DNA melting in aggregates: impeded or facilitated?. J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2005, 109: 13024-13029.Google Scholar
- Michoel T, Peer Van de Y: Helicoidal transfer matrix model for inhomogeneous DNA melting. Phys Rev E Stat Nonlin Soft Matter Phys. 2006, 73: 011908-Google Scholar
- Djuranovic D, Hartmann B: Conformational characteristics and correlations in crystal structures of nucleic acid oligonucleotides: evidence for sub-states. J Biomol Struct Dyn. 2003, 20: 771-788.Google Scholar
- Djuranovic D, Hartmann B: DNA fine structure and dynamics in crystals and in solution: the impact of BI/BII backbone conformations. Biopolymers. 2004, 73: 356-368. 10.1002/bip.10528.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.