Preparation of Well‐Defined DNA Samples for Reproducible Nanospectroscopic Measurements

Abstract

Due to its well-defined topology and chemical structure, DNA could become a biological standard sample in the field of nanospectroscopy. Tip-enhanced Raman spectroscopy (TERS) provides new insights into individual DNA molecules immobilized on flat mica crystals. The high sensitivity of TERS is used to assess the chemical changes that appear in DNA upon different surface immobilization protocols.

Structure and Properties of DNA Molecules Over The Full Range of Biologically Relevant Supercoiling States

“…Inverting this equation, we can calculate α from the contour length of DNA deposited on APTMS-treated mica (which is considered to be in the B form), and the length of DNA deposited in presence of MgCl 2 . From our results we obtain a value α = 0.4, in good agreement with the published value of 0.39 32 .…”

“…A further drawback of this technique is that the cation requirement limits the range of experimental conditions that can be used. Alternatively, treatment of mica with aminosilanes is also an effective procedure for nucleic acids binding; silanized mica enables a stronger binding of nucleic acids and tends to trap DNA molecules in a configuration that corresponds to the 2D projection of their solution conformation 31 , 32 ; however, hydrolysis of aminosilanes and aggregation is a complication associated with this technique.…”

“…In a recently published paper, Lipiec et al . 32 suggested that the local structure of DNA molecules deposited on aminosilane-treated mica resembles that of the B crystal form; in contrast, when molecules were deposited using the divalent cation adhesion method, a partial transition toward the A form occurs. If we take into account the structural parameters of the two DNA forms (A form: twist 32.7 degrees, rise 0.26 nm per base.…”

Tip‐Enhanced Raman Spectroscopy: A Tool for Nanoscale Chemical and Structural Characterization of Biomolecules

“…If DNA molecules are immobilized on a mica substrate with bridging by divalent cations (Mg 2+ or Mn 2+ ), AFM measurements reveal that they are shortened upon drying, which is consistent with a partial conformational transition from the B to the A form . This is corroborated by TERS through the disappearance of the signal assigned to the O−P−O stretching vibration at about 830 cm −1 , a small spectral shift towards high wavenumbers of the PO 2− symmetric stretching mode at about 1095 cm −1 , and the intensification and narrowing of the band assigned to the PO 2− asymmetric stretching mode at about 1250 cm −1 . Furthermore, local conformational changes seem to occur preferentially at the end of linear DNA molecules, according to the nanoscale TERS observation of a higher proportion of A‐form species at the extremities of DNA strands .…”

“…Much stronger DNA–surface bonding is indeed observed upon mica silanization than that with cation bridging, which hinders the B‐to‐A conformational transition and allows DNA to preserve its B form on the silanized substrate . Strong DNA–silane interactions can, however, affect the spectral signature of DNA and may obscure its description due to enhanced silane modes emerging only in DNA‐rich regions …”