Journal of Nanomaterials & Molecular NanotechnologyISSN: 2324-8777

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Research Article, J Nanomater Mol Nanotechnol Vol: 2 Issue: 7

Effect of Humidity on Structural Distortion and Conductance of DNA Nanowire

Ram Ajore1,2*, Inderpreet Kaur1, Harsimran Kaur1 and Lalit M Bharadwaj1
1Biomolecular Electronics and Nanotechnology Division, Central Scientific Instruments Organization, Chandigarh, India
2Department of Hematology, Lund University, Lund, Sweden
Corresponding author : Dr. Ram Ajore
Department of Hematology, Lund University, Lund, Sweden
Tel: 0046-2220735
E-mail: [email protected]
Received: November 06, 2013 Accepted: November 29, 2013 Published: December 03, 2013
Citation: Ajore R, Kaur I, Kaur H, Bharadwaj LM (2013) Effect of Humidity on Structural Distortion and Conductance of DNA Nanowire. J Nanomater Mol Nanotechnol 2:7. doi:10.4172/2324-8777.1000132

Abstract

Effect of Humidity on Structural Distortion and Conductance of DNA Nanowire

One of the intensively explored domains of the current bionanotechnology is the focus for search of nano-materials intended to develop high throughput electronic devices. Among the questioned physical materials deoxyribonucleic acid (DNA) has given promising background to be explored as potential nanowire material for aspiring nano-devices. The distinguished characteristics of electron hopping between DNA bases intrigues investigators to provide insights of the structrual properties of the DNA under varying relative humidity condition. Present manuscript attempts to provide insights for conductance of double stranded λ-DNA and its short stretch of intrinsic sequences in correspondence to structural distortion as a result of different relative humidity (RH) conditions.

Keywords:

Keywords

Nanomaterials; Electrical properties; DNA; Molecular electronics

Introduction

The basic branches of science Physics, Chemistry and Biology has instigated a number of sister branches laying down the foundation for the finest research outcomes touching different aspects of life. In today’s scenario, one of the most fascinating domains of biology is bio-nanotechnology with hope for the most intriguing interplay between physical and biological materials. Nature has devised numerous competent, finely tuned biological devices capable of carrying encoded information and decodes them into functionalities whenever needed. Indeed, the blue print of life, deoxyribonucleic acid (DNA), is the core biological material of great essence.
Since the discovery of DNA by Watson & Crick in 1950, it has attracted biologists for its wonderful capability of information coding and decoding property. From the physicist eyes, DNA is construed as an excellent physical material due to its π-electron rich base stacks. Studies on π-electron rich bases stack have embarked the assumption that DNA could be one of the promising nanowire for future molecular electronics [1]. A theoretical studies outcome unambiguously hints at the potential future of DNA in molecular electronics [2]. Photooxidation studies for charge transfer efficiency in DNA were carried out with different types of DNA binding and substitution agents [3]. Several factors correspondingly seem to underlying in controlling DNA conductance such as sequence length [4], DNA bases [5], humidity [6] etc. Literatures available on DNA conductance, aiming at humidity mainly emphasizes on water molecules availability on DNA polymer. Much less literature has been documented that renders the concern of DNA structure due to presence or absence of water molecules hence, affecting conductivity [7]. Minor structural distortion affects electronic coupling and hence DNA conductance was previously reported [8]. Concept of water molecules availability renders structural modification in DNA led us to experiment if this is related to DNA conductivity. In this study, we have tried to put together humidity with structural distortion effect insights on DNA conductance.
Concurrently, even a single base mutation in DNA may introduce significant changes in basic DNA structure. However, it is difficult to show direct correlation of single base change on spatial DNA structural modification. Hence, we attempted to show indirectly, the effect of structural distortion of DNA with respect to change in coupling energies, which is an important factor in determining the conductivity of DNA. This structural distortion mediated change in coupling energies, conclusively seems to be related with structural distortion introduced by lack of availability of water molecules in DNA. In DNA helix, base pair stacking is related to hydrophobic nature of bases. Presumably, removal of water molecules from the hydrophilic phosphate backbone will result in structural distortion in DNA helix due to unwinding (corresponding to base flipping) [7]. Flipping will result in sterical realignment of base atoms, which may affect the interaction of π-electron cloud of adjacent bases. In the present manuscript, conductance of double stranded λ-DNA and its intrinsic sequences under different condition has been studied. Experimental out comes have been explicated in terms of structural deformation or reformation in presence or absence of water molecules. Hence, to correlate the role of structural distortion for DNA conductance, electronic coupling energies were calculated by flipping bases. Structural distortion phenomenon due to humidity may add an important factor in determining the range and characteristics of DNA nanowire conductivity. It can also be helpful in determining the range of variability in DNA conductivity with respect to change in DNA bases.

Materials and Methods

Chemicals
Three sets of thiolated (5’ends) primers; Pr1 (1F-ATGCTTGAACCCGCCTATGC, 1R-TCACTTCATGCTTCGGCTTGAC), Pr2 (2F-TGGGATATTACGTCAGCGAGGAC, 2R-CACTTCATGCTTCGGCTTGAC) and Pr3 (3F-TGACTGCTGCTGCATT Introduction GACG, 3R-GCCATGATTACGCCAGTTGTAC) were procured from Bio Basics Inc., Canada. Thiolated primers were used to amplify intrinsic sequences (1.9, 2.9 and 3.9 kb) of λ-DNA. These sequences were immobilized by Dip and Drop approach [9] on in-lab fabricated electrodes of spacing 0.6, 1.0 and 1.3 μm [10]. The microelectrodes were fabricated using Optical Tweezer. Lambda-DNA was immobilized between electrodes of different spacing (5-25 μm) on an array of area 35×25 μm2 as mentioned earlier [9]. Enzymes and chemicals were of molecular biology grade and were procured from Q.BIO gene, USA; USBIOLOGICAL, USA; Banglore Genei Pvt. Ltd., INDIA; PIERCE, USA; BIO BASIC INC., Canada; Sigma Aldrich, USA. Solutions were prepared in deionized ultra pure water (ELGA Purelab ultra system, UK). In order to exclude the role of counter ion effect during I-V measurement, samples were prepared in deionized water. To avoid unstucking of bases of DNA and RNA, Magnesium and other biological inorganic ions were not added [11].

Results and Discussion

Amplification of thiolated DNA
Thiolated Fragments of 1910, 2947, 3970 bp (Figure 1) were amplified on MJ Research Gradient cycler (PTC-200) and were Purified using Nucleotrap polymerase chain reaction (PCR) Purification kit by BD Biosciences. In further description fragments 1910, 2947 and 3970 bp are represented as Pr1, Pr2 and Pr3, respectively.
Figure 1: Electrophoretically separated PCR amplified DNA fragments. M-marker, Lane 1-1.9, Lane 2-2.9 and Lane 3-3.9 bp fragments.
I-V Characterization
PCR synthesized and purified guanine-rich (Pr1, Pr2 and Pr3) and λ-DNA sequences were immobilized between microelectrodes and were electrically characterized under different environmental conditions.
I-V characterization under nitrogen controlled condition show current in the range of 10-12 A at 10 V in four consecutive measurements (Figure 2a). Hygro-thermometer was used for % RH measurement. Similar to vacuum measurement no current was observed in N2 controlled condition [12]. I-V Characteristics for Pr1 at 60% RH shows increase in current by a factor of 105 from dry condition (Inset Figure 2a). Controlled experiments were also performed in order to be sure that the conductivity observed was due to DNA excluding effects of impurities. Electrodes with immobilized DNA were treated in a solution containing DNase-I for a period of 30 minutes. Open circuit was observed on I-V characterization of DNase treated electrodes. This confirms I-V characterization of DNA only. The structure of DNA is thought to be randomly distributed between electrodes as isotropic electrical characteristic was observed.
Figure 2: I-V Characterization of Pr1, Pr2, Pr3 and λ-DNA. [a] under nitrogen ( Inset at 60% RH), [b] ambient and [c] 30% RH (Inset different % RH) condition.
Average of three measurements under ambient condition for all the four sequences is shown in Figure 2b. This shows an increase in current as compared to Figure 2a by a factor of 105. This increase in current at this stage suggests the pivotal role of mobile H+ ion. Hydronium ions (H3O+) are the dominant charge carriers at this stage.
It is revealed by infrared spectroscopy (IR) that five or six water molecules are coordinated to the oxygen atom of single phosphoric acid at 65% RH [13]. I-V curve was found to be destroying at 20% RH (Inset Figure 2c) but it was normal at or above 30% RH. Current measured at 30% RH shows non-linear behavior with a current range of 10-8 A (Figure 2c). This current range is 10 times less as compared to current measured at ambient condition. This ascertains DNA conducting nature and concurrently debars direct role of water molecule at 30% RH. Here, it is assumed that 30% RH is the optimum humidity where the number of water molecules are not enough to contribute in DNA conductance but sufficient to stabilize the structure of DNA helix.
Electronic coupling
In DNA helix base pair stacking is related to hydrophobic nature of bases. Removal of water molecules from the hydrophilic phosphate backbone will result in structural distortion in base stacking (corresponding to flipped base condition). Hence, to correlate the role of structural distortion in DNA conductance, electronic coupling energies was calculated by flipping the bases at middle and terminal positions in a random sequence of DNA. Electronic coupling between pairs of nucleic acid bases is a necessary ingredient in all models describing charge transfer in DNA. Electronic coupling energy was calculated using the single point calculations on the B3LYP/6-31G (d, p) geometry using the semi empirical intermediate neglect of differential overlap (INDO) Hamiltonian. The distance, r, between the base pairs i.e. the distance between double membered aromatic moieties in the third dimension is kept constant as in case of B-DNA is 3.38 Å such as close packing is avoided. Coupling energies were calculated using the energies of the HOMO, HOMO-1, of the stack of base pairs, obtained with the INDO Hamiltonian at the DFT/ B3LYP optimized geometry. The energy difference of the HOMO and HOMO-1 gives the energy splitting for hole transport.
Random DNA sequences considered G(A)5G(A)4G and G(A)3G(A)3G(A)2G) are of 12 base pairs length. In Figure 3a, middle and terminal end two bases of G(A)5G(A)4G were flipped. Terminal end bases (Guanine) were kept constant in Figure 3b. The spread of the values of the coupling of a given couple caused by slightly different geometry is remarkable. In particular, the spread is relatively large for couples flipped at terminal ends (Figure 3a). Almost same results were observed in Figure 3b. The outcomes can be further corroborated by the study of Sadowska-Aleksiejew et al. [8] where quantum mechanical calculations on the electronic coupling in dimers containing structurally distorted base pairs leads to change in hole transfer.
Figure 3: Electronic coupling in randomly selected DNA sequences. [a]-Coupling energy in DNA sequence with flipped middle and end bases. [b]-Coupling energy variation in DNA sequence with flipped middle bases.
Discussion
Presumably, DNA conductance under different environmental conditions shows varying nature of conductivity. Extremely low current measured in the range of 10-12 A at 10 V under nitrogen controlled condition (Figure 2a) has two fold basis either it is due to loss of water molecules which are suppose to be contributing in conduction [6] or due to the loss of threshold number of water molecules required to stabilize the base stack. Indeed, it was earlier postulated and established that the force required for the base pairs to stack on one another as fully as possible is provide by hydrophobic nature of bases.
DNA conductance measured at ambient condition (Figure 2b) is again having two fold bases either the conductance is due to the increased dielectric constant as a result of ions produced by the water molecules or due to effective base stacking due to presence of threshold number of water molecules. If first would have been the case the conductance of λ-DNA should characteristically differ from the conductance of other three sequences Pr1, Pr2 and Pr3 being comparatively longer molecule. Hence, it is pointed out that DNA conductance is through π-electron rich base stacks with its correct geometrical orientation.
Conductance measured for Pr1 at different % RH determines limit of threshold humidity to uphold base stacking in natural DNA (Inset Figure 2c). Conductance reduces and curve line shifts from the measured curve line at 30-40 % RH as the RH approaches 20% or less. Here, it is presume that the effect is due to unavailability of threshold number of water molecules required to stabilize the DNA bases stacking. Once again, it is pointed out that conductance observed at 30% RH (Figure 2c) excludes direct role of water molecules but it has role in base pair stacking in DNA helix. Current measured was in the range of 10-8 A at 10 V at 30% RH (Figure 2c). Current in ambient condition (Figure 2b) is 10 times more than at at 30% RH (Figure 2c) which indicates the role of excess water molecules (Hydronium ions) at ambient condition. Therefore, role of water molecules is in accordance to idea of stabilization DNA base pairs stacking and not for conductivity near 30% RH.
The study and discussion about the charge carriers and the effect of nucleobase electronic coupling may be insufficient to draw concrete conclusions about the structural distortion but is serves the purpose that little change in nucleobase position causes significant change in electronic coupling which is a necessary ingredient for charge transfer. This further strengthens result of DNA insulating behavior at N2 controlled condition due to utmost distorted DNA structure.

Conclusion

In the present study, we attempted to add insights of DNA conductivity under different environmental condition. The dominant role of water molecules in DNA conductance was found above 30% RH. Humidity below 30% results in reduced conductance and shifting of curve line from measured curve line at 30-40% RH. It is inferred from the experiment outcomes that current curve shift is due to lack of threshold number of water molecules required to up hold the base stacking necessary for DNA conductance. Hence, Lack of base stacking will disturb the naturally stable form of DNA model as established by Watson and Crick. On the other hand, trivial structural distortion with change of single base brings significant differences in electronic coupling energies of DNA bases, which is an important parameter determining electronic conductivity of DNA nanowire.
Insufficient availability of water molecules rendering structural distortion in DNA and single base mutation in DNA introducing significant changes in DNA structure as shown by coupling energy measurement suggest the important role of DNA structural in its conductivity. However, it is difficult to show direct correlation of single base change with structural change of DNA. Hence, we have indirectly shown the effect of structural distortion of DNA with respect to coupling energies which is an important factor in determining the conductivity of DNA. This structural distortion mediated change in coupling energy is conclusively relates with structural distortion introduced by lack of availability of water molecules in DNA as their results fall for the same concept of structural distortion. Overall, we infer that structural distortion is an essential parameter determining the conductivity of DNA nanowire. Structural distortion phenomenon due to humidity will add an important factor in determining the range and characteristics of DNA nanowire conductivity. Present report is an important addition to the scientific literature as it shows that the varying humidity conditions leads to variation in the DNA conductivity.

Acknowledgment

This work was supported by Department of Biotechnology and Department of Science and Technology. Authors are thankful Dr. Prakash from GETECH Hyderabad, India for microelectrode array fabrication. One of us authors (Ram Ajore) thanks Council of Scientific and Industrial Research, Delhi for providing fellowship for Ph.D.

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