Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification

Abstract

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

DNA Sequencing: Current State and Prospects of Development

“…The technique of reproducible conductivity measurements on single nucleotides with limited conformation and an improved algorithmic approach for isolating nucleotide bases is proposed in the [80]. The method of quantum point contact single-nucleotide conductance sequencing uses combed and electrostatically connected single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules.…”

“…It is shown that by changing the applied bias and the pH condition, it is possible to turn on and turn off the molecular conductivity, which leads to a reversible nucleotide perturbation for electronic recognition. In [80], the potential of using simple surface modifications and existing biochemical fragments in individual nucleotide bases for reliable, direct, single-molecular nanoelectronics DNA and a method for identifying nucleotides of RNA for sequencing is demonstrated.…”

High‐Throughput Block Optical DNA Sequence Identification

“…Samples were left to dry in air prior to analysis. See Figure S6 of the Supporting Information for DNA surface density discussion …”

“…See Figure S6 of the Supporting Information for DNA surface density discussion. [39] Multiplexed Imaging and Optical Vibrational Spectroscopy (Raman and FTIR): The Raman spectra of DNA, benzenethiol, and nucleotideglycine mixtures were acquired using a home-built confocal setup. The samples were imaged using an inverted Zeiss microscope with a 100× objective (NA of 0.85), and the light was focused on the entrance port of a triple grating Princeton Instrument imaging spectrophotometer (Acton SpectraPro SP-2500 equipped with a PIX100B-SF camera).…”

A machine learning approach for accurate and real-time DNA sequence identification

“…With the emergence of machine learning tools over the last decade, there is an increased interest in utilizing these methods to identify molecules from the current's signature. Recently, surfaceenhanced Raman spectroscopy [10], scanning tunneling spectroscopy (STS) [9,11,12], scanning tunneling microscopy break junction (STM-BJ) [13], and the measurement of ionic current blockade in nanopores [14] have been used to identify/sequence single-stranded DNA, RNA, and peptides. Identification of small molecules from a mixture was also recently demonstrated using machine learning classification methods [15][16][17].…”