Introduction of Physicochemical Properties Termed Stickiness and Pseudostickiness to Quantification of Macromolecule-interaction and Its Application to The Analysis of Lambda Genome DNA

Koichi NISHIGAKI and Yoshito SAKUMA

Elucidation of specific interactions of biological macromolecules is a central theme in molecular biology. Data based on precise analyses are accumulating for interactions of nucleic acid vs nucleic acid, protein vs protein, and nucleic acid vs protein. The contribution of dynamic and less stable structures on related functions is becoming more and more significant1). It is impossible, for example, to understand general PCR products without such consideration2). Inter- and intra-molecular structures, which have life-times comparable to the time-scale of biological reactions (e.g., enzyme-catalyzed DNA polymerization), were crucial in determining the final products2). The same dynamical feature of macromolecules was also operating in separating highly homologous single- stranded DNAs (e.g., single-nucleotide-substituted DNAs)3), which is a theoretical basis for the current popular technique, SSCP (single strand conformation polymorphism)4).
In this paper, we introduce a parameter which allows us to measure interactions of macromolecules, especially those of nucleic acids. The parameter can be called stickiness since it seems to be proportional to a residence-time, how sticky it is, of a ligand molecule within a local sphere through the movements of attaching and detaching between sites of a macromolecule. Then, we present a computer-aided calculation method for pseudostickiness which is manageable and still consistent with the above definition.
The application of the program to lambda genome DNA revealed that the spatial distribution of sticky sites for a certain oligonucleotide along lambda DNA is indicative of its gene arrangement. We also investigated the effect of single- and multiple-base-substitution of a primer DNA on stickiness to a template DNA. Through these computer experiments, the effectiveness of introducing a parameter stickiness or pseudostickiness to the analysis of macromolecules is supportively discussed.


Definition of Stickiness--- Stickiness can be introduced by purely theoretical considerations as shown in Fig.1a. It is a property inherent to an interaction between two kinds of molecules, a ligand (L) and an anti-ligand (A). Stickiness (s) can be defined as a relative residence- time of a ligand in a local sphere assigned on an anti-ligand as expressed in the equation 1.


where t(Pi, Pj) stands for a mean travelling time for ij or a mean binding time for i = j regarding the points of Pi and Pj, r is the ratio of the area of a local sphere to that of a unit sphere, n is the number of binding sites and t is an average unit-sphere-passing-through time in the medium of interest. P0 and Pn+1 equal Pin, entrance point, and Pout, exit point, respectively. It is evident that if there is no bonding between A and L at Pi, t(Pi, Pi) = 0 (perfect reflection) and if, in addition, a ligand is thrown back by a single contact with an anti-ligand at P1 ( a center of a local unit sphere) as shown in Fig.1b, s = 0 (no stickiness).
Originally, stickiness is defined for a local interaction just as

Fig. 1. Definition of stickiness.
(a) Stickiness is defined, as described in the text, through statistical ensemble of the residence times of a ligand (symbolized as B) in the inner and the outer regions of a unit sphere (broken-line circle) fixed in an anti-ligand (i.e., macromolecule) symbolized as A. In the inner part of a unit circle, the ligand is in the state of either binding at the points such as P1, P2 and P3 or travelling between those points and the residence time can be obtained from the times at Pin, entrance into the sphere, and Pout, exit out of the sphere. (b) Zero stickiness. The symbols are the same as those denoted in
Fig.1a. If no binding at P1 just as perfect reflection, stickiness is reduced to zero (see Eq.1). Free travelling in the medium is also zero stickiness by definition.

described above. However, it can be expanded to a global one by the following formulation:


where sg is the global stickiness of a pertinent anti-ligand molecule to a ligand and N is the total number of local spheres. We would like to treat this general stickiness in a more detailed manner elsewhere, which must have some relation to the equilibrium constant of binding and has an analogous nature of the frequency factor appearing in kinetic theory.
In this paper, we want to lead it to a tool for describing DNA-DNA interactions where an oligonucleotide is a ligand whereas a macromolecule DNA (often a template DNA) is an anti-ligand. Since it is difficult to define a local sphere exactly ( 3-D solution structures of DNA are not so well-known), we have to introduce some approximation for it. A primary way to substitute for stickiness is to use an equilibrium constant of binding or at a certain site of a template DNA, and we term it pseudostickiness. It can be so rationalized that since in Eq.1, t(Pi, Pi), mean staying time, is generally far greater than t(Pi, Pi+1), mean travelling time, the major factors that govern the value of stickiness, s, are staying times and by that the staying time has a close relation to an equilibrium constant of binding, which is the ratio of association to dissociation kinetic constant and is far greater than unity. In other

Fig. 2. Calculation of binding free energies as a substitution of stickiness for a DNA-DNA system. A binding free energy between a template (anti-ligand) and a primer (ligand) in a particular conformation at a certain local site of template DNA is computed by way of the conventional method based on the thermodynamical parameters for helix stability and non-helix (called as 'coiled part' in the figure) stability (see Ref.
5). All possible structures conformed to the constraints are subjected to the calculation of their binding free energies by computer and those structures which are more stable than a threshold stability ( in Table I) are picked up together with their free energies and the site- informations. The free energy values thus obtained are used as a substitution of stickiness or pseudostickiness at the site. As shown in the figure, the coiled parts consist of both bulge and internal loops, this being taken into consideration during their free energy calculation.

words, the greater the binding constant, the more the stickiness.
Secondly, we think that calculation time for searching stable structures between a template and a primer along the sequence of a template is a good candidate for emulating stickiness since it includes similar processes2) with attaching-and-detaching in the phenomenon defined above: the algorithm allows the intensive calculation along the region which conforms to the given requirements as shown in Table 1 and it leads to long local residence (long calculation times) in calculating stabilities of a DNA complex between a template and a primer along the template DNA sequence. The local residence time for calculation will become longer as the concentration of stable structures becomes higher in a limited region. Thus, the calculation time will be roughly proportional to the stickiness and can be another pseudostickiness. In the following section (RESULTS AND DISCUSSION), we will use -derived pseudostickiness rather than the calculation time-derived one.

Table I. Constraints imposed for searching stable structures and the parameters used in the program.


Computer Programming--- PASCAL was used as a programming language. The program, STICKINESS, which offers pseudostickiness of was derived from a PCR analysis program, PCRAna, which was made for searching stable and semi-stable hybrid structures between a template DNA and an oligonucleotide and then applied to working out possible PCR products2). The essential scheme for calculating the stability of a hybrid structure is depicted in Fig.2. The thermodynamic parameters used were those of Salser5), which were obtained for RNA but are known to be still usable for DNA6). The constraints imposed on hybrid structures are listed in Table I. Pseudostickiness expressed in terms of binding constant, , was directly used just as it was calculated by a computer without any transformations.
Experimental Procedures--- Programs were usually performed on the main- frame computer of the Computer Center of the University of Tokyo. The DNA selected for this experiment was that of a bacteriophage, lambda (48502 bp)7). For ligands, tens of oligonucleotides, including single-base- substituted ones, were selected as indicated in the legends to the figures. The positions at which stable hybrid structures were found were mapped on the circle of the lambda DNA sequence for each ligand (oligonucleotide).


Pseudostickiness applied to lambda genome DNA--- Various ligands of dodecadeoxyribonucleotide were used to examine the pseudostickiness of lambda DNA. The results for the ligands of a homo- or co-polymeric

Fig. 3. Pseudostickiness maps of the systems of homo- or co- polymeric oligonucleotides and lambda DNA. The oligonucleotides used were (a) d(ApApApApApApApApApApApA) or d(A)12 in abbreviation (hereafter, similar molecules are shown in this form), (b) d(T)12, (c) d(C)12, (d) d(G)12, (e) d(GT)6, (f) d(GA)6, (g) d(GC)6, (h) d(AT)6, (i) d(AC)6 and (j) d(TC)6 whereas the template was lambda DNA throughout. Bars were stacked at the 5'-end point for each hundred-nucleotide window sequence in proportion to the number of structures picked up within the corresponding region. The arrows indicate the origin in the lambda DNA map and the numbering proceeds clockwise (see Fig.4).

sequence are shown in Fig.3. Each ligand is shown to have characteristic preferential bindings or pseudostickiness to lambda DNA. From a viewpoint of genome structure, these results can be compared to those obtained with G+C content analysis and DNA stability analysis, which had been done by Wada and Suyama8). Those sticky regions depicted in Fig.3a are (A+T)- rich and also thermodynamically less stable regions according to their results, which are quite natural. These regions are especially remarkable because they fall on unique sites in lambda genome DNA: the left and the

Fig. 4. A genetic map of lambda DNA. The genes were assigned on lambda DNA together with a brief description of their function. The starting point of the map (cos site) is indicated by an arrow and the direction of numbering is the same (clockwise) as in

right ends of central region (map position; 23000 and 38000, respectively) as shown in Fig.4. This fact was not so clear in G+C content analysis or thermal stability analysis8). Furthermore, the ligand of dA12 has local pseudostickiness solely to central region, which embosses the region against the remaining ones, although dT12, another (A+T)-rich sequence have a less clear tendency. As Wada and Suyama pointed out, structural features disclosed by thermodynamical stability analysis have a stronger relationship to biological functions than those revealed by G+C content analysis which does not contain physical meanings8). Since the pseudostickiness analysis is based on thermodynamics, characteristic features revealed by it seem to have some connection with biological functions. It should be noted that (A+T)-rich (or (G+C)-rich) region analysis could not offer similar results to those provided by the pseudostickiness analysis with dA12, though it is not possible at present to predict, a priori, which ligand is effective to reveal such properties.
The other ligands have their characteristic stickiness patterns. This means that there are as many probes as the diversity of ligand sequence in order to characterize anti-ligand DNAs such as lambda genome DNA. This fact is further reinforced by the following experiment.
Single-base-substitution effect--- Five ligands which are single-base- substitutions of an oligonucleotide, pfM12 (dAGAACGCGCCTG), gave the pseudostickiness analysis result shown in Fig.5. It evidently indicates that even a single-base-substitution in a ligand sequence significantly affects the result of pseudostickiness. This fact is worthy of note since there are no differences in (G+C)-content between pfM12-8A and pfM12-8T or among pfM12-8C, pfM12-4C and pfM12-4G and therefore, the differences detected here are what (G+C)-rich region analysis can never detect. During these analyses, apparent differences should be translated with enough caution since the pseudostickiness, which is expressed by a frequency of bars in Fig.3 and Fig.5, can be changed by a cut-off parameter ( G) employed. However, we can make a safe comparison among the data if they are obtained under the same conditions as those given here.
Significance of stickiness--- In this paper, we coined the term, stickiness, together with its definition. Then, we expanded the concept of stickiness to a manageable one, pseudostickiness, for DNA-DNA interaction. The pseudostickiness thus introduced can be regarded as a kind of concept derived from binding affinity (or binding constant). The most outstanding difference between pseudostickiness and binding constant is that the former contains the concept of space while the latter does not. In real phenomena occurring both in vivo and in vitro, macromolecule interactions

Fig. 5. Oligonucleotide point mutation effect on pseudostickiness. Single-base-substituted derivatives of an oligonucleotide designated pfM12 (dAGAACGCGCCTG) were examined regarding their pseudostickiness with lambda DNA. The derivatized sequences are: (a) pfM12 (original), (b) pfM12-8C (i.e., the position substituted is the 8th and the base to which the original base (G) was substituted is C, and the following are expressed likewise), (c) pfM12-4C, (d) pfM12-4G, (e) pfM12-8A and (f) pfM12-8T. The maps are drawn in the same way as in Fig.3.

are affected by spatial factors such as size, location and orientation, which is the reason for the necessity of introducing stickiness.
Originally, stickiness is defined for a local event so that it should be called local stickiness. It implies that there are multiple sticky sites in a macromolecule which compete to bind a ligand. If one of these sites is a so-called specific site, i.e., the strongest binding site, the ligand binding to this site (specific binding) must be influenced kinetically and thermodynamically by the ligand binding to the rest. This means that we cannot correctly describe the phenomenon of specific binding without considering the other non-specific bindings. Therefore, global stickiness, which denotes the stickiness applied to an entire macromolecule, becomes a useful property to measure the behavior of a ligand/anti-ligand system. The conventional approaches which neglect the global stickiness effect to describe a specific binding are simplistic and incorrect in this sense. The ratio, in size, of a local region to an entire macromolecule is crucial in determining whether such a systemic consideration is required. This is evident especially in an interaction of a giant DNA and a protein (or an oligonucleotide), where the value of the ratio is very small. In the usual studies, only the interaction between the targeted site of a macromolecule and a ligand is considered. However, we take the remaining regions of a macromolecule into consideration by means of global stickiness in order to obtain a quantitative prediction for the behavior of a ligand/macromolecule system. One illustrative example for this is an interaction between a promoter buried in a big DNA and an RNA polymerase. Without the information provided by global stickiness, one will not be able to explain their behavior quantitatively provided with their dissociation constants and their concentrations in a cell. In other words, the behavior of a molecule is affected by not only its direct partner but also its surroundings. Elucidation of this fact and introduction of a reasonable method to deal with this is the most essential contribution of this paper to science.


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