About

Hashim M. Al-Hashimi, PhD, is the Roy and Diana Vagelos Professor of Biochemistry and Molecular Biophysics at Columbia University Irving Medical Center, where he also serves as Associate Dean for Biomedical Graduate Education. Born in Beirut, Lebanon, and having grown up across Greece, Italy, Jordan, and the UK, he completed his undergraduate studies in Chemistry at Imperial College London and earned his PhD in Biophysical Chemistry from Yale University. His research focuses on developing a deep, quantitative, and predictive understanding of cellular processes based on the fundamental behaviors of nucleic acids and their interactions with protein binding partners. Al-Hashimi's contributions include helping develop residual dipolar coupling methodology during his graduate studies, which revolutionized the study of protein structure and dynamics by NMR. As a postdoctoral fellow, he expanded these methods to nucleic acids. His group has discovered many of the ubiquitous motional modes underlying the biological activities of nucleic acids, with significant implications for drug discovery, genome stability, and cancer. His research involves combining NMR spectroscopy, computational modeling, optical melting experiments, and chemical probing to determine 3D dynamic ensembles of RNA and DNA molecules at atomic resolution. His work has reshaped structural biology by emphasizing the importance of dynamic ensembles in understanding biomolecular function, including DNA replication fidelity, RNA folding, and gene regulation. Al-Hashimi has co-founded Nymirum Inc to enable RNA-targeted drug discovery using RNA dynamics and has developed methods harnessing RNA dynamic ensembles to identify small molecule inhibitors of HIV-1 replication. He has trained over thirty-five graduate students and postdoctoral fellows from diverse backgrounds, many of whom now lead laboratories or work in biotech and pharmaceutical industries worldwide. His research continues to explore the role of nucleic acid dynamics in cellular processes, viral RNA function, mutagenesis, and cancer, employing high-throughput sequencing and other innovative approaches to map DNA structural dynamics genome-wide and to design targeted inhibitors.

Selected publications

• Assessing the contribution of rare DNA states to cancer mutational signatures using sequence-specific conformational fingerprinting

  Nature Communications · 2026-04-23

  articleOpen accessSenior author

  Rare and short-lived DNA conformations are proposed to be key drivers of mutagenesis, yet assessing their contribution to mutational signatures found in human cancers remains challenging. Here, we develop an approach that quantifies the sequence-dependent propensity to form a rare DNA conformation and compare the resulting fingerprint against cancer mutational signatures. Using 19F NMR, we measure the propensity for the anionic Watson-Crick-like G•T− conformation across all sixteen triplet sequence contexts and discover a striking 50-fold variation driven by suboptimal interactions between anionic thymine and its 3’ neighbor. Comparing this fingerprint, and those of other rare DNA states against the Catalogue of Somatic Mutations in Cancer (COSMIC) database uncovers plausible links to mutational processes associated with exposure to damaging agents and therapies. Thus, integrating molecular biophysics with genomic epidemiology provides a powerful framework to explore how DNA’s dynamic properties shape genome stability and influence human disease. In this work, researchers developed an approach to quantify how often DNA adopts a rare, mutagenic conformation, the anionic Watson–Crick-like G•T mismatch, across all possible sequence contexts. Using 19F NMR spectroscopy, they discovered that the likelihood of forming this state varies by up to 50-fold depending on neighboring bases.

  PublisherOA PDFDOI

• Probing rare and short-lived conformational states in nucleic acids using off-resonance carbonyl and guanidino carbon R1ρ relaxation dispersion

  Journal of Magnetic Resonance · 2025-05-29

  articleOpen accessSenior authorCorresponding
  PublisherOA PDFDOI

• Assessing the contribution of rare DNA states to cancer mutational signatures using sequence-specific conformational fingerprinting

  Research Square · 2025-11-19

  preprintOpen access1st authorCorresponding
  PublisherOA PDFDOI

• An A‐T Hoogsteen Base Pair in a Naked DNA Hairpin Motif: A Protein‐Recognized Conformation

  Angewandte Chemie · 2025-04-11

  articleSenior author

  Abstract In duplex DNA, A‐T and G‐C form Watson‐Crick base pairs, and Hoogsteen pairing only dominates upon protein binding or DNA damage. Using NMR, we show that an A‐T Hoogsteen base pair previously observed in crystal structures of transposon DNA hairpins bound to TnpA protein forms in solution even in the absence of TnpA. This Hoogsteen base pair, located adjacent to a dinucleotide apical loop, exists in dynamic equilibrium with a minor Watson‐Crick conformation (population ∼11% and lifetime ∼55 µs). Extending the apical loop to three residues inverted the equilibrium, making Watson‐Crick the dominant state and the Hoogsteen conformation recognized by TnpA a minor state (population ∼14% and lifetime ∼28 µs). The propensity for Hoogsteen pairing depended on apical loop residues, which form contacts directly or indirectly stabilizing the Hoogsteen conformation. A structure survey did not reveal Hoogsteen pairing near RNA apical loops making them unique to DNA. Our results demonstrate that Hoogsteen can be the dominant state even in naked unmodified duplex DNA and identify 5′‐CTT(T/C)AG‐3′ as a DNA‐specific apical loop motif stabilized by Hoogsteen pairing. Hoogsteen base pairs may be prevalent in DNA hairpins forming during replication and transcription, with broad implications for the genomic landscape.

  PublisherDOI

• Quantitative and Systematic NMR Measurements of Sequence-Dependent A–T Hoogsteen Dynamics in the DNA Double Helix

  Biochemistry · 2025-02-21 · 5 citations

  articleSenior authorCorresponding

  The dynamic properties of DNA depend on the sequence, providing an important source of sequence-specificity in biochemical reactions. However, comprehensively measuring how these dynamics vary with sequence is challenging, especially when they involve lowly populated and short-lived conformational states. Using 1H CEST supplemented by targeted 13C R1ρ NMR experiments, we quantitatively measured Watson–Crick to Hoogsteen dynamics for an A–T base pair in 13 trinucleotide sequence contexts. The Hoogsteen population and exchange rate varied 4-fold and 16-fold, respectively, and were dependent on both the 3′- and 5′-neighbors but only weakly dependent on monovalent ion concentration (25 versus 100 mM NaCl) and pH (6.8 versus 8.0). Flexible TA and CA dinucleotide steps exhibited the highest Hoogsteen populations, and their kinetics rates strongly depended on the 3′-neighbor. In contrast, the stiffer AA and GA steps had the lowest Hoogsteen population, and their kinetics were weakly dependent on the 3′-neighbor. The Hoogsteen lifetime was especially short when G–C neighbors flanked the A–T base pair. Our results uncover a unique conformational basis for sequence-specificity in the DNA double helix and establish the utility of NMR to quantitatively and comprehensively measure sequence-dependent DNA dynamics.

  PublisherDOI

• Probing Rare and Short-Lived Conformational States in Nucleic Acids Using Off-Resonance Carbonyl and Guanidino Carbon R1ρ Relaxation Dispersion

  SSRN Electronic Journal · 2025-01-01

  preprintOpen accessSenior author
  PublisherDOI

• Revealing hidden protonated conformational states in RNA dynamic ensembles

  Nucleic Acids Research · 2025-11-21 · 1 citations

  articleOpen accessSenior author

  Identifying protonated states within RNA ensembles, quantifying their pKas, and elucidating the kinetic mechanisms by which they form is essential for understanding protonation-coupled biochemical reactions and how RNAs sense and adapt to pH fluctuations. However, detecting protonated states is challenging when they are short-lived and lowly populated. Here, using pH-dependent NMR chemical exchange, kinetic solvent isotope effects, and mutation, we show that a low-populated (0.4% at pH 6.4) conformational state of HIV-1 TAR RNA is coupled to protonation of a C⁺-C mismatch. Despite an intrinsic pKa of ~7.1, the energetic penalty to form this alternative conformation depressed the apparent pKa to ∼4.0, below the pH range typically probed experimentally. Substituting C-C with a G-C base pair abolished the pH-dependence of these dynamics, confirming C-C as the protonation site. This hidden protonated state competes with a more abundant conformation harboring a C-A⁺ mismatch, producing a non-monotonic ensemble response to pH. Both transitions follow an induced-fit mechanism, in which solvent-exposed nucleobases are rapidly protonated followed by slower changes in secondary structure. These findings reveal a general mechanism for protonation-coupled conformational switching in RNA and provide a framework for dissecting sparsely populated protonated states and their multi-protonation-state dynamics.

  PublisherDOI

• Insight into the Conformational Ensembles Formed by U–U and T–T Mismatches in RNA and DNA Duplexes From a Structure-based Survey, NMR, and Molecular Dynamics Simulations

  Journal of Molecular Biology · 2025-05-08 · 3 citations

  articleSenior authorCorresponding
  PublisherDOI

• Kinetic Dissection of Proton-Coupled Conformational Transitions in Nucleic Acids by Integrating pH-Dependent NMR and Chemical Modifications

  Journal of the American Chemical Society · 2025-05-27 · 8 citations

  articleOpen accessSenior authorCorresponding

  Proton-coupled conformational transitions play fundamental roles in nucleic acid recognition, catalysis, and folding, yet the kinetic mechanisms underlying these multistep protonation reactions remain unknown. Here, we present an approach to resolve the dominant kinetic pathway and rate-limiting step, which combines NMR chemical exchange measurements with chemical perturbations that shift pKa or modulate conformational equilibria. Applying the approach to three nucleic acid systems, we find the microscopic protonation step to be a diffusion-limited proton transfer reaction (kprot ∼ 1011 M–1 s–1), 2 orders of magnitude faster than diffusion-limited ligand-binding. For an A+–C mismatch in duplex DNA, protonation was the rate-limiting step occurring after the conformational change at a diffusion-limited kon ∼ 1011 M–1 s–1 via conformational selection of the wobble conformation, which forms rapidly and in significant abundance in the neutral ensemble. In RNA, the A–C wobble was sparsely populated in the neutral ensemble. The apparent kon was 2 orders of magnitude slower, and the reaction followed an induced-fit mechanism, where the unpaired adenine was initially protonated, followed by rate-limiting intrahelical flipping. The apparent kon was 5 orders of magnitude slower for the protonated G(syn)–C+ Hoogsteen conformation in duplex DNA in which cytosine protonation was rate-limiting occurring after the conformational change via conformational selection of an energetically disfavored G(syn)–C intermediate. These kinetic models quantitatively predicted the impact of pH shifts and chemical modifications on reaction kinetics. Our findings reveal how differences in nucleic acid conformational ensembles can drive diverse kinetic responses to pH changes and chemical modifications, even in binding reactions involving the simplest ligand: the proton.

  PublisherOA PDFDOI

• An A‐T Hoogsteen Base Pair in a Naked DNA Hairpin Motif: A Protein‐Recognized Conformation

  Angewandte Chemie International Edition · 2025-04-11 · 1 citations

  articleOpen accessSenior authorCorresponding

  In duplex DNA, A-T and G-C form Watson-Crick base pairs, and Hoogsteen pairing only dominates upon protein binding or DNA damage. Using NMR, we show that an A-T Hoogsteen base pair previously observed in crystal structures of transposon DNA hairpins bound to TnpA protein forms in solution even in the absence of TnpA. This Hoogsteen base pair, located adjacent to a dinucleotide apical loop, exists in dynamic equilibrium with a minor Watson-Crick conformation (population ∼11% and lifetime ∼55 µs). Extending the apical loop to three residues inverted the equilibrium, making Watson-Crick the dominant state and the Hoogsteen conformation recognized by TnpA a minor state (population ∼14% and lifetime ∼28 µs). The propensity for Hoogsteen pairing depended on apical loop residues, which form contacts directly or indirectly stabilizing the Hoogsteen conformation. A structure survey did not reveal Hoogsteen pairing near RNA apical loops making them unique to DNA. Our results demonstrate that Hoogsteen can be the dominant state even in naked unmodified duplex DNA and identify 5'-CTT(T/C)AG-3' as a DNA-specific apical loop motif stabilized by Hoogsteen pairing. Hoogsteen base pairs may be prevalent in DNA hairpins forming during replication and transcription, with broad implications for the genomic landscape.

  PublisherOA PDFDOI

Labs

• Al-Hashimi LabPI

Awards & honors

• Roy and Diana Vagelos Distinguished Professorship (2022)
• Fellow of the Biophysical Society (2021)
• Fellow of the International Society of Magnetic Resonance (2…
• NAS Award in Molecular Biology (2020)
• James B. Duke Distinguished Professorship (2015)