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Department of Bioorganic Chemistry

Electrostatics in the Self-assembly of DNA and RNA

Turner and his coworkers were the first to realize the role of the nearest neighbor interactions in the self-organization and stabilization of the DNA and RNa duplexes. They also elucidated the free-energy contributions in the homo and hetero duplex stability owing to the  the nearest neighbor interactions. Readers are cited to the Turner Group's home page for their pioneering work.

We have however recently given concrete experimental evidences  through pKa  measurements (PDF 338, PDF 339, PDF 341, PDF 343, PDF 355, PDF 361) that the electrostatic interactions through the nearesst-neighbors play an important role in steering both intra and intermolecular forces culminating into the self-assembly of  both single-stranded and homo and hetero duplexes of DNA and RNA. We indeed could show that because of cross-modulation of the aromatic character (evidenced by  a change of pKa ) amongst the nearest-neighbors as a result of  electrostatic cross-talk, a clear pKa modulation take place. The generality of this pKa modulation by  electrostatic cross-talk was also clearly shown in a much simpler coupled system consisting of pyridine-benzene, covalently linked by linker, which was transformed to pyridinium-benzene  by simple change of the pH. The change of the aromatic character from pyridine to pyridinium was electrostatically registered by the phenyl group in the steric proximity by showing the  pKa modulation  [PDF 343]

Basis of  the above pKa modulation: Such electrostatic interactions is also expected to steer recognition and interactions of the ligand by the nucleic acid by the complementary electric field. The overall stability of nucleic acids, as well as their complexes with a ligand, is considered a sum of contributions of intermolecular electrostatic interactions, hydrogen bonding, van der Waals and hydrophobic interactions. ( PDF 338, PDF 339, PDF 341, PDF 343, PDF 355, PDF 361)  Measurable entity of the different electrostatic/stereoelectronic effects in nucleic acids and protein folding is pKa perturbation. Determination and interpretation of pKa of an ionizable group (a basic center for protonation or a dissociable group) in biomolecules highlights the molecular mechanism of its biological function. The shift in pKa  values is an important source of information about neighboring charges, electrostatics, structural perturbation as well as partial charge distribution over the whole molecule, and differential hydration of the microenvironment. Structural studies have revealed that in a large number of RNAs (and in certain cases in DNAs), the pKavalues of nucleobases (particularly adenine and cytosine) are significantly perturbed relative to that of mononucleotides. Thus, the most effective acid-base catalysis can be performed with ionizable groups having pKa near physiological pH of 7, thereby accounting for widespread use of histidines in protein enzyme active sites.

For reviews see Chapter 6 of Dr. Parag Acharya's Ph.D. Thesis. and Chapter 6 of Dr. Sandipta Acharya's Ph.D. thesis


(A) Electrostatic cross-modulation of the pseudoaromatic character in single-stranded RNA by nearest-neighbor interactions

The generation of a single anionic or cationic center at an alkaline or acidic pH in a given molecule presents a unique opportunity to examine the electrostatic make-up of these molecules both at the neutral or ionic state. The generation of a single cationic center in the phenyl-nicotinamide system provided new straightforward evidence showing that the charge density of the electron-deficient pyridinium was actually enhanced by the donation of the charge from the electron-rich phenyl group (i.e., the pyridinyl became more basic by ca. 0.5 pKa unit compared to an analogous system where phenyl was absent) owing to the electrostatic interactions between these two moieties. On the other hand, the generation of the 5'-guanylate ion in, for example, the hexameric single-strand (ss) RNA [5'-GAAAAC-3'], in comparison with the constituent trimeric [PDF 341], tetrameric, pentameric, and hexameric [PDF 344, 355]-ssRNAs, has unequivocally shown how far the electrostatic cross-talk (as an interplay of Coulombic attractive or repulsive forces) in this electronically coupled system propagates through the intervening pAp nucleotide steps until the terminal pC-3' residue in comparison with the neutral counterpart. The footprint of the propagation of this electrostatic cross-talk among the neighboring nucleobases is evident by measurement of pKas from the marker protons of ionization point (i.e., of G) as well as from the neighboring marker protons (i.e., of A or C) in the vicinity, as well as from the change of the chemical environment (i.e., chemical shifts) around their aromatic marker protons owing to a change of the stacking-destacking equilibrium as a function of pH.

Details are provided in References [PDF 355, 344, 341, 338] as well as in the Ph.D. Thesises of Dr. Parag Acharya and Dr. Sandipta Acharya
 

B) Propagation of electrostatic interplay across the single strand (Papers 344 and 347)

Perturbed pKa is not repulsive destacking. What I meant in my papers is that the influence of guanylate ion is most felt on the stacked nucleobases by electrostatics because they are stacked.and in proximity..... with what is written here..... "It has been observed that the propagation of the interplay of various electrostatic forces across the ssDNA chain is considerably weak, since the perturbed pKa is only NMR detectable effectively up to the fourth nucleobase moiety in the hexameric ssDNA (15c). In comparison, this perturbed pKa is observed, by NMR titration experiment, for the corresponding isosequential ssRNA up to the sixth nucleobase residue, which measures upto ~21 in the unfolded state. This suggests that the intramoleclar attractive stacking is weaker in the isosequential ssDNA than those of the ssRNA. Here, we are focussing our attention to the relative penaly of stacking in ssDNA versus ssRNA. That is to compare the N-type sugar/ with pseudoaxial nucleobase in ssRNA versus S-type sugar with pseudoequatorial nucleobsae in ssDNA, in one hand, visavis more hydrophobic environment in ssDNA versus hydrophilic 2'-OH group in ssRNA compared to ssDNA. In this competition, if we assume that generation of a gradient of negative charge in guanylate ion will electrostatically influence by an influx of negative charges to those nuclebases which are most stacked and meaning they are in the orbital-overlapping proximity. Then, the stronger stacking in ssRNA, as evident by its most perturbed pKas of the constituent nucleobases, will clearly mean that the N-type sugar with pseudoaxial nucleobase in ssRNA is more stabilizing than the constituent hydrophilic 2'-OH group in each pentofuranose sugar [PDF 355]

Figure 1. Electrostatic Interplay across GpA1pA2pA3pA4pC

Figure 2. pKa perturbations from nearest-neighbor interactions
 

(C) Base pairing versus stacking contribution in overall RNA-RNA and DNA-DNA duplex stabilization - a qualitative understanding from pKa measurement  of model monomeric nucleotides (Paper 347 )

Figure 3. Watson-Crick Base Pairing
 

Because we can successfully measure the pKa of guanine-9-yl from either of the aglycones in the RNA timers 3 and 4,it shows that the aglycones in the trimeric RNAs constitute a coupled heterocyclic system right across the pH range, 6.7 to 11.5 owing to both 3'->5' and 5'->3' two-way cross-modulation by electrostatic interaction. This may be the reason a trimeric RNA sequence constitutes a single codon signal in recognition and function in the protein synthesis machinery. The magnitude of the chemical shift change in any of the aromatic protons in either of the two coupled aglycones differs in a variable manner depending upon the geometry of stacking, partial charge of the heteroatom as well as the sequence
(compare GpApA and GpApC), which is evident from relative chemical shift change.

(1) Increasing content of A-T base pairs weakens the stability of DNA-DNA duplex over the corresponding RNA-RNA duplexes
(2) Strength of stacking of A-T rich DNA-DNA sequence [due to CH3 (1-Thyminyl) -p (of 9-adeninyl) interaction] increases compared to A-U rich sequence in RNA-RNA duplexes

Conclusions (Paper 347 ):

(1) The nucleobases of the monomeric DNA are uniformly more basic than the corresponding RNA counterparts.

(2) The strength of the base-pairing based on the pKa difference (DpKa) of the monomeric donor and acceptor can be used to understand the relative base-pairing strength of larger oligomeric DNA-DNA and RNA-RNA duplexes.

(3) The use of DpKa among the monomer blocks, modeling the A-T/U and G-C base pairs, allows us to understand the base-pairing contributions in the free energy of DNA-DNA and RNA-RNA duplex stability, which is evident from a high correlation coefficient found between
D37 of the helix stability and the sum of the DpKa values of donor/acceptor in the base-pair formation.

(4) The high correlation of D37  of the helix stability and the sum of DpKa values in a duplex showed that a simple subtraction of the base-pairing contribution of RNA-RNA over DNA-DNA ([Dbp ]RR-DD) from the free energy of the total helix stability ([D37 ]RR-DD) gives us the relative stacking contribution ([Dstacking ]RR-DDin a qualitative manner.
 
 
 

Figure 4. Free energy stabilization of the base-pairing ([Dbp ]RR-DD) [red triangles, R = 0.84] as well as that for stacking ([[Dbp ]RR-DD) [blue squares, R = 0.85] in RNA-RNA (RR) over the DNA-DNA (DD) duplex as a function of %A-T/U bp content in the duplexes. (R - correlation coefficient).

(5) A comparison of these two linear plots ([Dbp ]RR-DD) versus  ([Dstacking ]RR-DD)  as a function of % A-T/U bp content, Figure 4)
with opposite slope shows that with the increasing content of A-T base pairs the stability of DNA-DNA duplex weakens over the corresponding RNA-RNA duplexes ([Dbp ]RR-DD) while the strength of stacking  ([Dstacking ]RR-DD) of A-T rich DNA-DNA sequence increases in comparison with A-U rich sequence in RNA-RNA duplexes. This increased stacking contribution from T compared to U, in DNA-DNA over RNA-RNA duplex, comes from favorable electrostatic CH/s interaction between the 5-methyl group of T with the nearest-neighbor A in the AT rich sequence.

See References [PDF 344, 347] for details.
 

(D) Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds (Paper 361 )
 

We here show that the electronic properties and the chemical reactivities of the internucleotidic phosphates in the heptameric ssRNAs are dissimilar in a sequence-specific manner because of their non-identical microenvironments,in contrast with the corresponding isosequential ssDNAs.This has been evidenced by monitoring the dH8(G) shifts upon pH-dependent ionization of the central 9-guaninyl (G ) to the 9-guanylate ion (G-),and its electrostatic effect on each of the internucleotidic phosphate anions,as measured from the resultant d31P shifts in the isosequential heptameric ssRNAs vis-`a-vis ssDNAs. These oligos with single ionizable G in the centre were chosen [ PDF 361] because of the fact that the pseudoaromatic character of G can be easily modulated in a pH-dependent manner by its transformation to G- (the 2'-OH to 2'-O-  ionization effect is not detectable below pH 11.6 as evident from the N1-Me-G analog), thereby modulating/titrating the nature of the electrostatic interactions of G to G- with the phosphates,which therefore constitute simple models to interrogate how the variable pseudoaromatic characters of nucleobases under different sequence context (J. Am. Chem. Soc., 2004, 126, 8674-8681) can actually influence the reactivity of the internucleotide phosphates as a result of modulation of sequence context-specific electrostatic interactions. The physico-chemical studies  [ PDF 361]  have shown that same specific p2 ,p3 and p4 phosphates in the native heptameric RNAs are more prone to the alkaline hydrolysis because of their relatively enhanced electrophilic character resulting from weaker 31P screening. This screening effect originates as a result of the systematic charge repulsion effect between the electron cloud in the outermost orbitals of phosphorus and the central guanylate ion, leading to delocalization of the phosphorus p-p charge into its d-p orbitals. It is thus likely that, just as in
the non-enzymatic hydrolysis,the enzymatic hydrolysis of a specific phosphate in RNA by general base-catalysis in RNA-cleaving proteins (RNase A, RNA  phosphodiesterase or nuclease) can potentially be electrostatically influenced by tuning the transient charge on the nucleobase in the steric proximity or as a result of specific sequence context owing to nearest-neighbor interactions. [ PDF 361]

See References [PDF 361] for details.
 
 

(E) Extended Genetic Code (Paper 341 )

The net result of this electrostatic cross-talk between two neighboring aglycones as a result of base-base stacking is creation of a unique set of aglycones in an oligo or polynucleotide, whose physico-chemical properties are completely dependent upon the nearest neighbor electrostatic interactions. This has considerable implication in the specific ligand binding ability, aptamer recognition, RNA catalysis, and most probably in codon-anticodon interaction. The poorer pseudoaromatic cross-modulation and stronger stacking in ssDNA compared to ssRNA (as manifested in their respective pKas) may have considerable implications in serving the purpose of DNA as a carrier of the genetic code (its almost error-free replication and transcription). On the other hand, the conformational flexibility of ssRNA allows it to create different scaffolds and nascent folded states with the characteristic tunable dielectrics, giving variable microenvironments (resulting in to larger pKa variation), which is manifested in its its dual
biological role in general, as we witness in the translation machninery and catalysis.

Thus in a RNA sequence, P1 Q1 N Q2 P2 , the actual physico-chemical integrity of N is dictated by the pseudoaromatic character of both neighboring Q1 and Q2 , whose properties are further tuned by the electronic nature of  P1 and P2. Hence, the relative stacking-destacking in any two adjacent nucleotides will actually set the ON and OFF switch for the tunability of the pseudoaromatic character of a particular nucleobase, N. Thus, the pseudoaromatic character of N can have at least 24numbers of variations, depending upon the chemical nature of the neighboring Q1 and Q2, which therefore implies that a given nucleobase sequence in a polynucleotide chain constitutes an unique extended genetic code, which can be turned ON and OFF depending upon the intrinsic dynamics of folding and unfolding within the molecule owing to the sequence context or interaction
with an external ligand.

See References [PDF 341] for details.
 
 

Related publications

355. Electrostatic cross-modulation of the pseudoaromatic character in single-stranded RNA by nearest-neighbor interactions.
 Pure and Applied Chemistry, 77 (1), 291-311 (2005).

349. Acharya, S.; Barman, J.; Cheruku, P.; Chatterjee, S.; Acharya, P.; Isaksson, J. and Chattopadhyaya, J. Significant pKa perturbation of Nucleobases Is an Intrinsic Property of the Sequence Context in DNA and RNA. J. Am. Chem. Soc. 2004, 126, 8674-8681.

347.  Measurement of Nucleobase p Ka Values in Model Mononucleotides Shows RNA-RNA Duplexes To Be More Stable than DNA-DNA Duplexes. Acharya, P.; Cheruku, P.; Chatterjee, S.; Acharya, S. and Chattopadhyaya, J. J. Am. Chem. Soc; 126, 2862-2869 (2004).

344. Acharya, P.; Acharya, S.; Amirkhanov, N. V.; Cheruku, P.; Földesi, A. and Chattopadhyaya, J. Cross-modulation of the pKa of Nucleobases in a Single Stranded Hexameric-RNA Due to Tandem Electrostatic Nearest-neighbor Interactions. J. Am. Chem. Soc. 2003, 125, 9948-9961.

341. Acharya, P.; Acharya, S.; Földesi, A. and Chattopadhyaya, J. Tandem Electrostatic Effect From the First to the Third Aglycon in the Trimeric RNA Owing to the Nearest-neighbor Stacking. J. Am. Chem. Soc. 2003, 125, 2094-2100.

343. Acharya, P.; Plashkevych, O.; Morita, C.; Yamada, S. and Chattopadhyaya, J. A Repertoire of Pyridinium-Phenyl-Methyl Cross-Talk through a Cascade of Intramolecular Electrostatic Interactions. J. Org. Chem. 2003, 68, 1529-1538.

339. Acharya, S.; Acharya, P.; Földesi, A. and Chattopadhyaya, J. Cross-Modulation of Physicochemical Character of Aglycones in Dinucleoside (3'->5') monophosphates by the Nearest Neighbor Interaction in the Stacked State. J. Am. Chem. Soc. 2002, 124, 13722-13730.

331. Acharya, P. and Chattopadhyaya, J. The Hydrogen Bonding and Hydration of 2'-OH in Adenosine and Adenosine 3'-ethylphosphate. J. Org. Chem. 2002, 67, 1852-1865.

301. Acharya, P.; Trifonova, A.; Thibaudeau, C.; Földesi, A. and Chattopadhyaya, J. The Transmission of the Electronic Character of Guanin-9-yl Drives the Sugar-phosphate Backbone Torsions in Guanosine 3',5'-bisphosphate. Angew Chem. Int. Ed. 1999, 38, 3645-3650.

361. Non-identical Chemical Characters of the Internucleotidic Phosphates can Modulate the Non-enzymatic Reactivity of the Phosphodiester Bonds in RNA. J. Barman, S. Acharya, S. Chatterjee, Å. Engström and J. Chattopadhyaya, Org. Biomol. Chem, 4, 928-941 (2006).