Measurements of Single Nucleotide Electronic States as

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Measurements of Single Nucleotide Electronic States as Nanoelectronic Fingerprints for Identification of DNA Nucleobases, their Protonated and Unprotonated States, Isomers, and Tautomers Josep Casamada Ribot, Anushree Chatterjee, and Prashant Nagpal J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b01403 • Publication Date (Web): 20 Mar 2015 Downloaded from http://pubs.acs.org on March 22, 2015

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The Journal of Physical Chemistry

Measurements of Single Nucleotide Electronic States

as

Nanoelectronic

Fingerprints

for

Identification of DNA Nucleobases, their Protonated and Unprotonated States, Isomers, and Tautomers Josep Casamada-Ribot,1 Anushree Chatterjee,1,2,3* Prashant Nagpal1,2,3,4* 1

Department of Chemical and Biological Engineering, University of Colorado, Boulder

2

Renewable and Sustainable Energy Institute, University of Colorado, Boulder

3

BioFrontiers Institute, University of Colorado, Boulder

4

Materials Science and Engineering, University of Colorado, Boulder

*Corresponding Author. ABSTRACT. Several nanoelectronic techniques have been explored to distinguish the sequence of nucleic acids in DNA macromolecules. Identification of unique electronic signatures using nanopore conductance, tunneling spectroscopy or other nanoelectronic techniques depends on electronic states of the DNA nucleotides. While several experimental and computational studies have focused on interaction of nucleobases with different substrates, effect of nucleic acid biochemistry on its electronic properties has been largely unexplored. Here, we present correlated measurements of frontier molecular orbitals and higher order electronic states for four DNA nucleobases (Adenine, Cytosine, Thymine, and Guanine), and first principle quantum 1 Environment ACS Paragon Plus


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chemical density functional theoretical (DFT) computations. Using different pH conditions in our experiments, we show that small changes in the biochemical state of these nucleic acids strongly affect the intrinsic electronic structure, measured using scanning tunneling spectroscopy (STS). In our experimental measurements and computations, significant differences were observed between the position of frontier orbitals and higher energy states between protonated and unprotonated nucleic acids, isomers, and different keto-enol tautomer’s formed in these nucleotides, leading to their facile identification. Furthermore, we show unique “electronic fingerprints” for all nucleotides (A, G, T, C) using STS, with most distinct states identified at acidic pH. These results can have important implications for identification of nucleic acid sequences in DNA molecules using a high-throughput nanoelectronic identification technique.

KEYWORDS DNA nanotechnology, biophysics, electronic states, tunneling spectroscopy, computational modeling TEXT. Nanoelectronic identification of DNA nucleotides is an important candidate for the next generation sequencing technologies, as it is an enzyme-free, high-throughput technique which does not require synthetic amplification, thus reducing the sample preparation time, cost, as well as number of random sequencing errors.1-12 Unique electronic signatures for different DNA nucleotides depends on differences in electronic states of the four nucleobases, which enables distinguishing them using conductance, tunneling or other nanoscale electronic measurements. Several studies have shown nanopore conductance of DNA nucleotides based on either ionic current change along the pore,3-7or tunneling current decay8 when a base is traversing the pore. While these systems have shown promising results, the high error rates result due to noise

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convoluted with signals, requirement of heavy bioinformatics analysis, as well as knowledge of the sequence (i.e. from other sequencing platform) to align the obtained data, limits their application for de novo sequencing.3,4 While some studies have used scanning tunneling microscopy and spectroscopy to study tunneling spectra of double-stranded and single-stranded DNA,9-12 lack of insight into the change in electronic states with changes in biochemical state of these nucleotides has prevented further progress in identification of easily distinguishable unique electronic signatures for each DNA nucleobase (Adenine (A), Thymine (T), Guanine (G) and Cytosine (C)). Here, we present detailed measurements of occupied and unoccupied molecular orbitals (frontier and higher energy electronic states) for the four DNA nucleobases (A,T,G,C) at different pH conditions. As the pH is varied for different nucleic acids, different protonated and unprotonated biochemical species, different isomers, and keto-enol tautomers are formed. Using correlated experimental tunneling spectroscopy measurements and first-principle quantum chemical DFT computations, we show that small changes in the biochemical state of these nucleic acids strongly affect their electronic structure. We also report distinctly observable differences in our measurements between the position of frontier orbitals and higher energy states, and facile identification of signatures for these protonated/unprotonated acids, isomers, and tautomers. This study provides valuable insights into the relationship between biochemical structure and electronic states of single molecules, and enables development of high-throughput DNA sequencing method for facile identification of sequence of nucleic acids and their biochemical state. Measurements of electronic density of states (DOS) of different homo-oligonucleotides were made on an atomically flat, clean Au (111) surface. DNA oligomers were drop casted after surface treatment of gold, and the desired pH was adjusted to obtain the nucleic acids in different



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biochemical states (see full methods in supporting information). Using nanoelectronic tunneling measurements of electrons and holes through single nucleotides of different nucleobases (A,T,G,C), we obtained the positions of the frontier molecular orbitals (lowest unoccupied molecular orbital (LUMO), highest occupied molecular orbital (HOMO)) and other higher energy electronic states (Figure 1a,b).13–15 Furthermore, we utilized DFT analysis of electronic states of nucleic acids16–19 to characterize the different eigenvalues or energies of the molecular orbitals and the electronic wavefunction to obtain valuable insights into the shape of electronic molecular orbitals for each nucleobase, either isolated20 or on a surface.19 All DFT simulations were performed on GAMESS software package21 using restricted Hartree-Fock method (see Methods in Supporting Information). To provide a better description for charged molecules22,23 or nucleobases in different biochemical states, we selected 6-311++G(2d,2p) basis set, which is suitable for charged species as it is split-valence triple zeta description of the Gaussian orbitals which also includes diffuse functions on both hydrogens and heavy atoms. We used a Becke 3parameter hybrid (B3YLP) density functional. While most of the computational focus so far has been on trying to understand and optimize the device-molecule configuration (i.e. change pore geometry or substrate metal),17,19 we investigated the DNA biochemistry and how it can affect the electronic properties of different nucleobases in various biochemical states. Using correlated experimental and computation investigations of these electronic states, we identified unique electronic states as “fingerprints” for facile identification of DNA nucleobases, unprotonated and protonated species, isomers, and other tautomer’s formed, with single nucleobase resolution. The experimental measurements of the electronic density of states (DOS) using tunneling spectroscopy13–15 were compared against computations of each nucleobase, which provided insights into the shifts in single nucleotide electronic states with changes in biochemistry and


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identification of an optimal biochemical state to differentiate between all nucleobases. A previous study showed significant differences in the electronic structure of purines and pyrimidines due to differences in their conjugation,15 which may also be exploited to identify individual nucleotides for DNA sequencing using tunneling spectroscopy. Furthermore, several biochemical changes in nucleic acids alter the electronic properties of DNA, eg. pH,24 with most nucleobases protonated at low pH values (below their pKa). We hypothesize that these changes can lead to measurable differences between the neutral and the protonated species, as previously reported by Florian et. al. using the IR and Raman spectra compared to ab initio calculations.25 Other biochemical changes may happen at low pH where tautomerization equilibria is driven towards enolic species, for instance for thymine.26–29 Therefore, these studies provide design rules for exploiting the biochemistry of nucleic acids for DNA sequencing. We explored the electronic states of different protonated and de-protonated nucleobases (using respective pKa for different nucleobases24). For cytosine protonated (pKa of 4.4) at N3 position (Figure 1c, unrestricted DFT studies predicts a protonation on the O-2 position forming an enol compound27,30), the DFT results show a significant change on both the frontier orbital wavefunction (Figure 1d) as well as on the different molecular orbital eigenvalues (or energies) when compared to the unprotonated cytosine (Figure 1c). This effect causes an increase of the HOMO/LUMO gap by ~0.4 eV on protonation, as well as a different distribution of ground and excited state electronic orbitals (Figure 1e). To verify this effect experimentally we performed STS measurements on single homopolymer cytosine nucleotides (Figure 2a,b). Indeed, we observed a significant increase in the cytosine HOMO/LUMO gap under acidic conditions (pH

Measurements of Single Nucleotide Electronic States as

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