Nucleic acids, molecules essential for all life, can adopt many alternative structures besides the well-known right-handed double helix, some of which have been reported to exist and function in vivo. One of the most appropriate methods for structural studies of nucleic acids is circular dichroism spectroscopy, utilizing structure-induced chirality due to the asymmetric winding of absorbing nucleobases. Using electronic CD and absorption spectroscopies in combination with melting experiments, we analyzed a conformational equilibrium between DNA double helix and two alternative conformations of nucleic acids, cytosine i-motifs and guanine quadruplexes, as a function of the primary structure of model G/C-rich sequences, containing blocks of G and C runs in particular DNA strands. This paper is a part of special issue dedicated to 70th anniversary of the Biophysical Institute of the Czech Academy of Sciences, where circular dichroism spectroscopy of nucleic acids has been used successfully and impactfully for many years.

• Keywords
• Circular dichroism spectroscopy, Conformation equilibrium, Cytosine i-motif, DNA, Guanine quadruplex,
• Publication type
• Journal Article MeSH

Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.

• MeSH
• Cell Cycle * MeSH
• Protein Conformation MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy methods   MeSH
• Proteins * chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteins * MeSH

I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

• MeSH
• Azides * MeSH
• Benzazepines * MeSH
• DNA MeSH
• HeLa Cells MeSH
• Humans MeSH
• Magnetic Resonance Imaging * MeSH
• Antibodies MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 7-iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine MeSH Browser
• Azides * MeSH
• Benzazepines * MeSH
• DNA MeSH
• Antibodies MeSH