Isotope-Edited Variable Temperature Infrared Spectroscopy for Measuring Transition Temperatures of Single A-T Watson-Crick Base Pairs in DNA Duplexes.

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2═O stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this "sub-molecular scale thermometer", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is ∼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL ∼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is ∼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL ∼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.

Modeling the Infrared Spectroscopy of Oligonucleotides with 13C Isotope Labels.

The carbonyl stretching modes have been widely used in linear and two-dimensional infrared (IR) spectroscopy to probe the conformation, interaction, and biological functions of nucleic acids. However, due to their universal appearance in nucleobases, the IR absorption bands of nucleic acids are often highly congested in the 1600-1800 cm-1 region. Following the fruitful applications in proteins, 13C isotope labels have been introduced to the IR measurements of oligonucleotides to reveal their site-specific structural fluctuations and hydrogen bonding conditions. In this work, we combine recently developed frequency and coupling maps to develop a theoretical strategy that models the IR spectra of oligonucleotides with 13C labels directly from molecular dynamics simulations. We apply the theoretical method to nucleoside 5'-monophosphates and DNA double helices and demonstrate how elements of the vibrational Hamiltonian determine the spectral features and their changes upon isotope labeling. Using the double helices as examples, we show that the calculated IR spectra are in good agreement with experiments and the 13C isotope labeling technique can potentially be applied to characterize the stacking configurations and secondary structures of nucleic acids.

Measuring the Enthalpy of an Individual Hydrogen Bond in a DNA Duplex with Nucleobase Isotope Editing and Variable-Temperature Infrared Spectroscopy.

The level of interest in probing the strength of noncovalent interactions in DNA duplexes is high, as these weak forces dictate the range of suprastructures the double helix adopts under different conditions, in turn directly impacting the biological functions and industrial applications of duplexes that require making and breaking them to access the genetic code. However, few experimental tools can measure these weak forces embedded within large biological suprastructures in the native solution environment. Here, we develop experimental methods for detecting the presence of a single noncovalent interaction [a hydrogen bond (H-bond)] within a large DNA duplex in solution and measure its formation enthalpy (ΔHf). We report that introduction of a H-bond into the TC2═O group from the noncanonical nucleobase 2-aminopurine produces an expected decrease ∼10 ± 0.76 cm-1 (from ∼1720 cm-1 in Watson-Crick to ∼1710 cm-1 in 2-aminopurine), which correlates with an enthalpy of ∼0.93 ± 0.066 kcal/mol for this interaction.

Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair.

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.