Protonation and Deprotonation of RNA Bases
by
Antarip Halder, Dhananjay Bhattacharyya, Abhijit Mitra
in
International Conference On "Nucleic Acids in Disease Disorder"
(NADD 2011)
Report No: IIIT/TR/2011/-1
Centre for Computational Natural Sciences and Bioinformatics
International Institute of Information Technology
Hyderabad - 500 032, INDIA
December 2011
Protonation and Deprotonation of RNA Bases
±,
±
Antarip Halder *, Abhijit Mitra , Dhananjay Bhattacharya
†
±
Center for Computational Natural Sciences and Bioinformatics (CCNSB), International Institute of
Information Technology (IIIT-H) Gachibowli, Hyderabad 500032
†
Biophysics Division, Saha Institute of Nuclear Physics (SINP), 1/AF, Bidhannagar, Kolkata 700064
Introduction
Results
Protonation of bases is important for both structure and function of nucleic acids :
Nucleobases get protonated/deprotonated mediated by environmental conditions (presence of metal ion 1,2,
presence of ligand3, pH of the medium4,5 or local pH modification etc).
Free energy changes of the protonation/deprotonation processes were studied for the isodesmic reaction;
Deprotonation:
Protonation:
Possible Protonation/Deprotonation Sites Of Individual RNA Bases.
Protonation sites are of two types, Class I and Class II
(I) Members of Class I take part in H-bond formation and provide proper electrostatic environment to
facilate catalytic processes e.g. oxyanion hole formation during peptidyl transfer reaction.6

Protonated bases lead to non-canonical base pairing in DNA7 and RNA8.

This class of protonated base pairs were first detected by BPFIND14 from observation of
proximity of two electronegative atoms of the bases. In absence of protonation these atoms
may repel each other while a proton attached to one of them lead to formation of a strong
hydrogen bond.
Protonation At Imino Nitrogen
Atoms Of Adenosine & Guanosine
(II)Members of Class II do not take part in H-bond formation but they are known to participate in proton
transfer processes.9

Important catalytic contributions to ribozyme activity is made by the protonated/deprotonated
states of nucleobases present in functional RNA.
Protonated base pairs in functional RNA:
Functionally important class of base pairs.
• Participate in tertiary interaction connecting distant structural elements.
• Nucleate higher order structures.
• Found in functionally important regions:
 Ribozyme cleavage sites, Protein recognition sites, other RNA motifs.
Extended Conjugation Due To Protonation At N3 of Guanine
Interaction Energy
= -33.26 kcal/mol
Protonation At Secondary Amino Nitrogen Causes
Bond Breakage.
Cahnges In Geometry Of Guanine Due To
Protonation (At N3) And Deprotonation (From N1)
Stability Of RNA Structure Through Protonated Basepair formation : The
Protonation Hypothesis. [Chawla et al. 2011]
The Larger Question
The question of energetics:
In RNA and DNA, the nucleobases typically exist as neutral species at biological pH (~7.4).
The average pKa1 values for protonated RNA bases are of the order ~3.2 (Guanine), ~4.1 (Adenine), ~4.4
(Cytosine) and the the average pKa2 values for neutral RNA bases are of the order ~9.3 (Guanine and
Uracil) 10,11.
It is not energitically favourable to have protonated RNA bases under physiological conditions.
Therefore, protonation/deprotonation of RNA bases must have some contribution to the global stability.
Geometry Optimization Of Possible Basepairs Containing N7Protonated Guanine, Resulted In The Transfer Of Proton From
N7 Of Guanine To The Nearest Electro-negative Atom Of The
Partner Base.
Changes In Geometry And Charge
Distribution Due To Protonation At
N7 of Guanine
Discussions
Protonation at side chains and imino nitrogens and deprotonation from imino or primary amino
nitrogen give relaxed stable geometry. Whereas, protonation at secondary amino nitrogen leads to
non-planar geometry on optimization at gas phase.
Protonation causes changes in geometry which makes it easier for the base to take part in noncovalent interactions.
Protonation at imino nitrogens is comparatively easier process.
Stable protonated base pairs (with interaction energy as low as ~20-40 kcal/mol) with imino notrogen
protonated Adenine(N1,N3), Cytosine(N3) and Guanine(N3) have been reported earlier 8.
Objective
To investigate,
How much thermodynamically 'unfavorable' is the process of protonation/deprotonation of
nucleobases.
After protonation/deprotonation what are the possible changes in geometry of a nucleo base
monomer which can contribute to increase the stability in the global context
Methods
Model Building:
– RNA monomers with neutral bases are taken from standard crystal structures of RNA.
Gas Phase Optimization and Energy Calculation:
– Quantum chemical calculations performed by GAMESS12 and GAUSSIAN0313 using different
levels of theory and approximation (B3LYP/6-31++G(2d,2p) and mp2/aug-ccpVDZ) to optimize
the geometry of neutral, protonated and deprotonated bases in gas phase at 298.15 K and 1 atm
pressure
Acknowledgment: DB and AM acknowledge partial support from
DBT project No. BT/PR-11429/BID/07/272/2008
Although protonation at N7 of Guanine is the least costly process, reduction in the acceptor potential
of its O6 atom constitutes a far stronger factor preventing N7 protonated guanine from participating
in base pair formation with its hoogsteen edge.
References
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