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RESEARCH

Ultrasensitive electrical detection of minute amounts of nucleic acid in bio-samples.

 

    We have demonstrated (Nature Nanotechnology (2020) 15, 836-840) that DNA containing a single-stranded fragment cannot conduct electrical current while a double helical DNA molecule can conduct electricity. That is because a precise arrangement of the bases (achieved in the double-stranded form) is required for the conductivity. Based on this observation, a detection scheme (illustrated in Fig. 1) was proposed. In the scheme hybridization of a nucleic acid molecule (either DNA or RNA) complementary to the linear fragment in the middle of a double-stranded DNA attached to electrodes arranges the bases in the single-stranded middle fragment in the double helical fashion, leading to electron flow through the DNA bridge.

    The electrical detection is extremely sensitive and we hope that the method will enable us to detect extremely low (up to 10^-20 M) concentrations of nucleic acid molecules in bio-samples.

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Fig. 1. - Schematic illustration of electrical detection of nucleic acid molecules. The DNA molecule is connected to two metal electrodes (dark orange triangles) through two gold nanoparticles covalently attached to its ends. The DNA is composed of two double stranded fragments at the ends and a single stranded one in the middle. The bases in the single stranded fragments are shown in different colours. No current is flowing through the circuit (A). When a DNA or RNA molecule containing a fragment (in blue) complementary to middle fragment of the DNA is getting captured through canonical base-pairing, the current in the circuit starts to flow (B).

Studies of conjugates between DNA and silver ions.

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     We have discovered that silver ions bind tightly to a double-stranded poly(GC)-poly(CG) DNA (see Fig. 3) composed of thousands of base pairs at stoichiometry of one Ag+ per GC pair. The binding causes movement of the strands one against another by one nucleotide such that in the resulting conjugate silver ions are positioned between CC and GG base pairs in the core of the molecule. The conjugate is, thus stabilized by Ag+ interaction with either CC or GG base pairs (C- Ag+-C and G- Ag+-G) in contrast to the initial poly(GC)-poly(CG) molecule stabilized by hydrogen bonds formed between G and C bases of the complimentary strands (Fig 3). In the conjugate, the ions are forming a chain in the very core of the molecule. The chain of red/ox active silver ions can strongly promote electrical conductivity of the DNA. We will study the conductive behavior of this and other similar molecules in collaboration with Prof. Danny Porath from Hebrew University of Jerusalem.

Reduction of Ag-ions in the conjugate (by BH4 or other chemical reductants) leads to formation of silver clusters in the DNA (step 2 in Fig. 3). Silver atoms, in contrast to the ions can’t stabilize CC and GG pairs. As a result, the initial (GC) base pairing in the DNA molecule is been restored. Since silver atoms, in contrast to the ions, are apolar they are located in the DNA core. Oxidation by molecular oxygen results in partial oxidation of the clusters. This partly oxidized conjugates are composed of a mixture of silver atoms and ions (step 3 in Fig. 3) organized in small clusters spaced by Ag-ions. This partly oxidized conjugate is fluorescent and characterized by emission at about 700 nm (excitation is at 560 nm).

  The fluorescent and conductive properties of poly(GC)-poly(CG)-silver conjugates will be studied. We are going also to investigate binding of silver ions with G4-DNA composed of four G-strands of equal length (short and long) and study electrical and fluorescent properties of the molecules.

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Fig. 3. – Scheme of Ag+ binding to poly(GC)-poly(CG). Incubation of the DNA with stoichiometric amount of silver ions (one Ag+ per GC base pair) yields stable conjugate between the nucleic acid and the cation (gray sphere). The strands of the DNA initially bound to each another by multiple of hydrogen bonds between G and C bases (three per each GC pair) are now bound through Ag+ ions. Reduction by borohydride, BH4 (reaction 2) results in formation of silver clusters (depicted in violet) in the core of the DNA molecule. Partial oxidation of sliver atoms by molecular oxygen   yields a conjugate capable of emit light at about 700 nm (being excited at about 600 nm)

Selective eradication of cancer cells by oxygen radicals (ROS) generated by ultrasmall gold nanoparticles

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     We have synthesized small (about 2 nm in diameter) gold nanoparticles which are, in contrast to commercial ones, capable of NADH oxidation by molecular oxygen. The oxygen molecule is reduced to an oxygen radical (Fig. 4A). These nanoparticles were incorporated into liposomes and delivered to cancer cells inside the vesicles. To target the liposomes in cancer cells antibody mimicking protein – DARPin_9-29 capable of specific and high-affinity interaction with HER2 receptors (overexpressed in breast cancer cells) were attached to the surface of the vesicles. We have demonstrated that the particles strongly reduce the viability of cancer cells and do not affect the healthy ones. The mechanism of the particle's effect on the cells and the involvement of reactive oxygen species (ROS) in the process are under investigation in collaboration with the laboratory of Prof. Sergey Deyev from the Institute of Bioorganic Chemistry (Moscow, Russia). 

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Fig. 4. A – Small gold nanoparticles composed of about 100 gold atoms (orange spheres) are catalyzing NADH oxidation by molecular oxygen. B – The particles being delivered to cells are killing them presumably by generating oxygen radicals during the oxidation of endogenous NADH. 
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