Tip-enhanced Raman spectroscopy of single RNA strands: towards a novel direct-sequencing method.

The sequencing of DNA or proteins is procedurally complex and requires sophisticated analytical techniques. DNA sequencing while a very powerful method requires separation and visualization methods to recognize specific DNA fragments. Furthermore, all the established methods require substantial amounts of DNA and fail to directly read the base composition of the strand. A method that utilizes the inherent information of the distinct bases present in DNA or RNA without the need of further labeling is therefore desirable. Recent approaches in this direction include pulling single DNA strands through nanopores, detecting certain electric properties, and then deducing the sequence. Attempts were also made to directly sequence DNA by using a scanning tunneling microscope. The main challenge with STM methods is always the low contrast and usually a statistic approach is necessary to evaluate the data. Our studies demonstrate that by using near-field optical techniques in combination with vibrational spectroscopy gives high contrast and the direct identification of bases on a single isolated RNA strand becomes feasible. Standard Raman spectroscopy makes the identification of the base components straight forward, however, the lateral resolution and the sensitivity of the method are far from the single-strand or even single-base detection levels required for a sequencing method. Herein we show that tip-enhanced Raman scattering (TERS) provides several advantages over conventional Raman spectroscopy: in just a few seconds acquisition time it gives high sensitivity at lateral resolution down to a few tens of nucleobases. These properties allow TERS mapping along a poly(cytosine) RNA strand. The results demonstrate the potential of the method to identify and sequence the composition of polymeric biomacromolecules (DNA, RNA, peptides). TERS spectra of a single-stranded RNA homopolymer of cytosine (poly(C)) have been measured with a spatial resolution down to a few nucleobases. The basic experiment is shown in Figure 1. A standard transmission TERS setup is used to focus a laser onto a silver-coated atomic-force microscope (AFM) tip, while the sample is moved independently, the sample surface is thus always in focus. Similar arrangements have been used to enhance infrared or Raman spectra of nanoscale materials, such as polymer samples, molecular monolayers, or single carbon nanotubes. In Figure 2 the topography image of a single-stranded RNA cytosine homopolymer is shown. To avoid Raman scattering from compounds other than RNA, the use of buffer solutions and other chemicals was kept to a minimum. However, as a Figure 1. A) The tip-enhanced Raman scattering (TERS) experiment along a single strand of RNA. B) Higher magnification of the area approximately corresponding to the size of the laser spot. C) Magnification corresponding to the interaction area of the TERS probe tip.