Summary of "Week 6 - Lecture 27"

Summary of “Week 6 - Lecture 27”

This lecture focuses on the application of Nuclear Magnetic Resonance (NMR) spectroscopy to study DNA and RNA structures, particularly analyzing proton chemical shifts and using 1D and 2D NMR techniques to investigate nucleic acid conformations, base pairing, and thermal stability.

Main Ideas and Concepts

1. Proton Chemical Shifts in DNA and RNA

Proton chemical shifts range from 0 to 15 ppm, typically referenced to TSP in water.
Different types of protons in nucleic acids include:
- Sugar ring protons: 1′, 2′, 2″, 3′, 4′, 5′, 5″.
- Base protons: H2, H8 (in guanine and adenine), H6 (in cytosine).
- Amino (NH₂) and imino (NH) protons: Present in bases G, C, A, T, and U.
Amino protons (NH₂) appear around 7.5 ppm; imino protons appear between 10–15 ppm and are involved in hydrogen bonding in base pairs.
Exchangeable protons (amino and imino) require recording spectra in H₂O to observe; non-exchangeable protons (attached to carbons) can be observed in D₂O.

2. Differences Between DNA and RNA Proton Shifts

RNA lacks 2″ protons due to the presence of a 2′-OH group, causing shifts and spectral crowding in the sugar proton region.
DNA shows more distinct chemical shift regions for sugar protons compared to RNA.

3. 1D NMR Spectra of Nucleic Acids

Typical 1D spectra show distinct regions for:
- Base protons (H6, H8, H2)
- Sugar protons (H1′, H3′, H4′, H5′)
- Methyl protons (from thymine)
Amino and imino protons appear in a separate downfield region (10–15 ppm).
Methyl groups from thymine show distinct peaks, aiding in sequence identification.

4. Thermal Stability and Melting Studies Using NMR

Monitoring imino proton signals at different temperatures allows observation of DNA duplex melting.
Base pairs in the interior of the duplex are more stable and retain signals at higher temperatures, while terminal base pairs melt first.
Melting temperature (Tm) is determined by plotting normalized chemical shifts of imino protons versus temperature, producing a sigmoidal curve.
Modifications in DNA (e.g., replacing oxygen with sulfur in phosphate groups) affect melting temperature and stability.

5. 2D NMR Spectroscopy – NOESY (Nuclear Overhauser Effect Spectroscopy)

2D NOESY spectra enable identification of individual protons in nucleic acids by revealing spatial proton-proton interactions.
Cross-peaks indicate short distances (<5 Å) between protons, useful for mapping base stacking and sugar-base interactions.
Sequential “walks” through the spectrum allow assignment of protons from one nucleotide to the next, providing information on nucleotide sequence and conformation.
NOE intensities are inversely proportional to the sixth power of the distance between protons, making distance estimation critical.
Different sugar conformations (anti vs. syn) affect observed NOE patterns.
Detailed analysis of NOESY spectra allows differentiation of self (intra-nucleotide) and sequential (inter-nucleotide) cross-peaks, confirming nucleotide order and structure.

6. Examples and Applications

Analysis of DNA hairpins and duplexes using imino proton signals.
Identification of terminal versus internal base pairs based on melting behavior.
Use of NOESY to assign protons in a DNA quadruplex-forming sequence.
Impact of chemical modifications on DNA stability assessed by NMR.

Methodology / Instructions Presented

Recording NMR Spectra of Nucleic Acids

Use TSP as chemical shift reference.
Record spectra in H₂O to observe exchangeable protons (imino and amino).
Use D₂O for non-exchangeable proton observation.

Analyzing 1D NMR Spectra

Identify sugar ring protons (1′, 2′, 2″, etc.).
Locate base protons (H2, H6, H8).
Identify methyl protons from thymine.
Monitor imino proton signals (10–15 ppm) for base pairing.

Thermal Melting Studies

Record spectra at multiple temperatures (e.g., 5°C to 60°C).
Observe disappearance of imino proton signals as duplex melts.
Plot normalized chemical shift intensity vs. temperature to determine melting temperature (Tm).
Compare Tm for modified vs. unmodified DNA to assess stability effects.

2D NOESY Spectroscopy

Collect 2D NOESY spectra to detect spatial proton-proton interactions.
Identify diagonal peaks (self) and cross-peaks (sequential).
Perform sequential walk by following base-to-base and base-to-sugar correlations.
Use NOE intensities and cross-peak patterns to assign nucleotide proton resonances.
Differentiate sugar conformations based on observed NOEs.

Speakers / Sources Featured

Primary Speaker: The lecturer delivering the content on NMR applications in nucleic acid structural analysis (name not provided).
No other speakers or external sources explicitly mentioned.

This lecture provides a comprehensive overview of how NMR spectroscopy, especially 1D and 2D NOESY, is used to study DNA/RNA structure, base pairing, and thermal stability, emphasizing proton chemical shifts and spectral interpretation strategies.