Probing the Hydrogen-Bonding Environment of Individual Bases in DNA Duplexes with Isotope-Edited Infrared Spectroscopy

Abstract

Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2=O carbonyl. We use solvatochromatic studies to show that the TC2=O signal’s position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2=O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH···O H-bond between adenine and thymine in both Watson–Crick and Hoogsteen base pairs in DNA.

The Local Vibrational Mode Theory and Its Place in the Vibrational Spectroscopy Arena

“…101 High delocalization and, consequently, different types of interactions between functional groups (vibrational coupling) make it hard to determine specific bond contributions as spectral signatures of polyatomic molecules of interest. 170,179 LMA offers a helping hand to detangle the congested information. 2D IR derived normal-mode frequencies suggest that specific CC and CO stretching vibrations in adenine (A), guanine (G), cytosine (C), and thymine (T) are prone to base pair coupling in the fingerprint region.…”

“…Linear and nonlinear vibrational spectroscopies have been of particular interest to monitor changes in the secondary structure of DNA. − Nevertheless, categorical structural assignment of the characteristic normal vibrational modes within the frequency range of 1500–1800 cm –1 , which is the fingerprint region of in-plane base vibrations, is hindered by spectral congestion. ,, Not only does spectral congestion pose a conundrum for nucleic acids, but for polyatomic molecules as a whole, as a result of the very nature of normal vibrational modes . High delocalization and, consequently, different types of interactions between functional groups (vibrational coupling) make it hard to determine specific bond contributions as spectral signatures of polyatomic molecules of interest. , LMA offers a helping hand to detangle the congested information.…”

“…Current and planned applied work in progress include the application of LMA’s unique features to declutter vibrational spectra, such as assisting isotope-edited IR spectroscopy pinpointing specific bonds of interest in the IR spectrum of DNA, which is hampered by the large number of overlapping carbonyl signals; monitoring oxidation state changes in photoactive enzymes via changes in LMA properties in support of, e.g., protein film electrochemistry; combining LMA with the group’s unified reaction valley approach (URVA) to model properties of artificial metalloenzymes and their catalytic mechanisms; , applying LMA’s potential in the emerging field of covalent binder drugs, which strongly depends on reliable, quantitative bond strength descriptors; , and on the other end of the spectrum, investigating a large number of reported ionic crystals with the far-reaching goal to compile a database with individual local mode force constants, serving as a basis for comprehensive studies on how important physicochemical properties, e.g., electrical conductivity and optical properties of these crystals are related to the individual bond strengths in these materials.…”

Modeling the Infrared Spectroscopy of Oligonucleotides with 13C Isotope Labels

“…1,3,23 With the successful applications of 13 C and 13 C� 18 O isotope labeling in the IR experiments of proteins to probe their folding, aggregation, and structural fluctuations, 24−34 researchers are extending these techniques to nucleic acids to elucidate their structure and dynamics in complex environments. 35,36 For example, in combination with 13 C and 15 N isotope labels, 2D IR spectroscopy was exploited to identify the stable structural motif and local disorders of parallel-stranded G-quadruplexes. 35 Recently, Stelling, Kreutz, and co-workers introduced the first example of single-atom 13 C labels into the C 2 position of thymine phosphoramidites, the monomer building blocks used to synthesize DNA oligomers.…”

Automatic Generation of Local Vibrational Mode Parameters: From Small to Large Molecules and QM/MM Systems

“…Vibrational spectroscopy can reveal in-depth information on the electronic structure and bonding of chemical systems, where its applications and improvements have been of recent interest. − Nevertheless, the very nature of a normal vibrational mode (NVM) in polyatomic systems is generally marked by delocalization in the form of collective motion of fragments, which makes it difficult to extract intrinsic bond properties and/or assigning contributions of a particular molecular fragment to a specific normal vibrational mode. The local vibrational mode (LVM) theory, , originally introduced by Konkoli and Cremer is a powerful tool to approach these complications by deriving local vibrations and associated local mode properties from NVMs, and to provide the foundation for the characterization of normal mode (CNM) procedure and the adiabatic connection scheme (ACS). − …”