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Published Work

2024/7/17 - MF Nüesch, L Pietrek, ED Holmstrom*, D Nettels, V van Roten, R Kronenberg-Tenga, O Medalia, G Hummer*, B Schuler*

The conformational dynamics of single-stranded nucleic acids are fundamental for nucleic acid folding and function.  Here, we employ a combination of single-molecule FRET, nanosecond FCS, fluorescence lifetime analysis, and nanophotonic enhancement to determine the conformational ensembles and rapid chain dynamics of short single-stranded nucleic acids in solution. To interpret the experimental results in terms of end-to-end distance dynamics, we utilize the hierarchical chain growth approach, simple polymer models, and refinement with Bayesian inference of ensembles to generate structural ensembles that closely align with the experimental data. Our results provide a detailed view of the conformational distributions and rapid dynamics of single-stranded nucleic acids.

2024/6/4 - SD Sperstand, ED Holmstrom*

The 3ʹ end of the hepatitis C virus genome is terminated by a highly conserved, 98-nucleotide
sequence called 3′X. This untranslated structural element is thought to regulate several
essential RNA-dependent processes associated with infection. 3′X has two proposed
conformations comprised of either three- or two stem-loop structures that result from different
base pairing interactions within the first 55 nucleotides. Here, we used single-molecule FRET
spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate
this region of the RNA (3′X55).

2024/1/16 - S Akhter, Z Tang, J Wang, M Haboro, ED Holmstrom, J Wang*, Y Miao*

Studying RNA-ligand interactions and quantifying their binding thermodynamics and kinetics are of particular relevance in the field of drug discovery. Here, we combined biochemical binding assays and accelerated molecular simulations to investigate ligand binding and dissociation in structured RNAs using the theophylline-binding RNA as a model system.

2023/9/29 - S Nepal, ED Holmstrom*

The hepatitis C virus (HCV) core protein (HCVcp) is the most highly conserved protein encoded by the HCV genome, and its N-terminal domain plays a crucial role in nucleocapsid assembly. Here, we use single-molecule FRET spectroscopy to study the energetic aspects of binding interactions involving HCVcp and antiviral therapeutics based on antibodies, aptamers, peptides, and small molecules. Our findings highlight distinct binding mechanisms associated with these molecular interactions.

2022/7/17 - S Sen, ED Holmstrom*

Nucleic acid-based drugs offer numerous opportunities for both academic and industrial endeavors. In this chapter, we describe how single-molecule FRET can be used to study aptamer-induced conformational changes within the nucleocapsid domain of the hepatitis C virus core protein.

2021/9/8 - SM Gunawardhana, ED Holmstrom*

Here, we use a series of aqueous-organic cosolvent mixtures to systematically modulate the solvent polarity around two different RNA-folding constructs that can form either secondary or tertiary structural elements. We show that the unfolded conformations of both model RNAs become more compact in apolar environments characterized by dielectric constants less than that of pure water. In the case of tertiary structure formation, this compaction also gives rise to more energetically favorable folding.

2021/7/20 - Z Tang, S Akhter, A Ramprasad, X Wang, M Reibarkh, J Wang, S Aryal, SS Thota, J Zhao, JT Douglas, P Gao, ED Holmstrom, Y Miao*, J Wang*

In this report, we show that a known small-molecule RNA splicing modulator (SMN-C2) binds to a single-stranded GA-rich sequence associated with exon 7 of the SMN2 pre-mRNA. Our findings suggest that the binding interaction is both sequence and sugar specific. Using information from NMR, computational simulations, and several structure-activity studies, we propose that this RNA sequence selectively binds the splicing modulator by forming a compact ligand-binding pocket comprised of two GAAG loops.

2021/2/13 - JC Sanders, ED Holmstrom*

Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins.

2021/1/13 - YMO Alhammad, MM Kashipathy, A Roy, JP Gagné, P McDonald, P Gao, L Nonfoux, KP Battaile, DK Johnson, ED Holmstrom, GG Poirier, S Lovell, AR Fehr*

Here, we report the crystal structure of the first macrodomain (Mac1) within nonstructural protein 3 (nsp3) of SARS-CoV-2. Mac 1 binds to ADP-ribose with low micromolar affinity and, like the macrodomains of other coronaviruses, enzymatically removes mono-ADP-ribose from target proteins containing this post-translational modification.

2020/9/18 - R Sharma, S KK, ED Holmstrom*, F Westerlund* 

Using a recently developed nanofluidic device to study the protein-induced compaction of genome-length nucleic acids, we observed that the hepatitis C virus core protein (HCVcp) initiates this process from the ends of these freely suspended polymers.

2020/2/2 - B Schuler*, A Borgia, MB Borgia, PO Heidarsson, ED Holmstrom, D Nettels, A Sottini

Here, we summarize the insights that have emerged from the highly disordered polyelectrolyte complexes identified so far, and we highlight recent developments and future challenges in (i) establishing models for the underlying highly dynamic structural ensembles, (ii) understanding the novel binding mechanisms associated with them, and (iii) identifying the functional consequences.

2019/6/5 - ED Holmstrom*, Z Liu, D Nettels, RB Best, B Schuler*

RNA chaperones are proteins that aid in the folding of nucleic acids, but remarkably, many of these proteins are intrinsically disordered. How can these proteins function without a well-defined three-dimensional structure?

2018/11/9 - F Sturzenegger, F Zosel, ED Holmstrom, KJ Buholzer, DE Makarov, D Nettels, B Schuler

The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex.

2018/10/4 - KK Grotz, MF Nueesch, ED Holmstrom, M Heinz, LS Stelzl, B Schuler, G Hummer

We combine single-molecule Förster resonance energy transfer (single-molecule FRET) experiments with extensive all-atom molecular dynamics (MD) simulations (>100 μs) to characterize the conformational ensembles of single-stranded (ss) DNA and RNA in solution.

2018/8/4 - NF Dupuis, ED Holmstrom, DJ Nesbitt

In this work, single-molecule Förster resonance energy transfer (FRET) confocal spectroscopy is used to study viscosity-dependent folding kinetics of an isolated RNA tertiary motif, that of the GAAA tetraloop receptor, allowing both solvent and internal frictional contributions to be investigated and extracted independently

2018/8/3 - ED Holmstrom, D Nettels, B Schuler

We have used single-molecule Förster Resonance Energy Transfer techniques to study the conformational dimensions and dynamics of the HCVcp nucleocapsid domain (HCVncd) at various stages during the RNA-induced formation of nucleocapsid-like particles.

2018/4/12 - Y Liu, ED Holmstrom, P Yu, K Tan, X Zuo, DJ Nesbitt, R Sousa, JR Stagno, Y Wang

Here, we present a step-by-step protocol for the solid–liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C 15 N-labeled segment;(ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues.

2018/1/1 - ED Holmstrom, A Holla, W Zheng, D Nettels, RB Best, B Schuler

Here we describe two essential elements of the quantitative analysis of single-molecule FRET data of IDPs: thesample-specific calibration of the single-molecule instrument that is required for determining accurate transfer efficiencies and the use of state-of-the-art methods for inferring accurate distance distributions from these transfer efficiencies.

2016/5/27 - ED Holmstrom, DJ Nesbitt

This review provides an overview of various temperature-dependent smFRET approaches from our laboratory and others, highlighting efforts in which such methods have been successfully applied to studies of single-molecule nucleic acid folding.

2016/5/3 - JT Polaski, ED Holmstrom, DJ Nesbitt, RT Batey

Using a combination of cell-based, biochemical, and biophysical techniques, we reveal the specific RNA architectural features enabling a cobalamin-dependent hairpin loop docking interaction between receptor and regulatory domains.

2015/12/15 - M Vieweger, ED Holmstrom, DJ Nesbitt

In this work, folding dynamics for the TLS domain of Brome Mosaic Virus have been investigated using single-molecule fluorescence resonance energy transfer techniques.

2015/5/4 - Y Liu, ED Holmstrom, J Zhang, P Yu, J Wang, MA Dyba, D Chen, J Ying, S Lockett, DJ Nesbitt, AR Ferré-D’Amaré, R Sousa, JR Stagno, Y Wang

Here we develop a hybrid solid–liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling.

2015/2/18 - ED Holmstrom, NF Dupuis, DJ Nesbitt

In the present work, we exploit single-molecule FRET (smFRET) techniques to measure, for the first time, the kinetic origins of these osmolyte-induced changes to the folding free energy.

2014/11/19 - ED Holmstrom, JT Polaski, RT Batey, DJ Nesbitt

Single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure. The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context.

2014/6/10 - NF Dupuis, ED Holmstrom, DJ Nesbitt

The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop–receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy.

2014/3/28 - ED Holmstrom, DJ Nesbitt

Here we use a minimal version of the human telomerase RNA pseudoknot to study this hairpin–pseudoknot structural equilibrium using temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) experiments.

2014/1/7 - ED Holmstrom, NF Dupuis, DJ Nesbitt

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10 pL) volumes of water.

2013/8/6 - NF Dupuis, ED Holmstrom, DJ Nesbitt

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level.

2012/10/19 - JL Fiore, ED Holmstrom, LR Fiegland, JH Hodak, DJ Nesbitt

In this work, single-molecule FRET (fluorescence resonance energy transfer) techniques are exploited to isolate and explore the cation-concentration‐dependent kinetics for formation of a ubiquitous RNA tertiary interaction, that is, the docking/undocking of a GAAA tetraloop with its 11‐nt receptor.

2012/4/23 - ED Holmstrom, JL Fiore, DJ Nesbitt

This work provides an analysis of how RNA–cation interactions affect the entropy and enthalpy associated with an RNA tertiary transition. Specifically, temperature-dependent single-molecule fluorescence resonance energy transfer studies have been exploited to determine the free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of folding for an isolated tetraloop–receptor tertiary interaction as a function of Na+ concentration.

2012/2/21 - JL Fiore, ED Holmstrom, DJ Nesbitt

This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop−receptor tertiary interaction as a function of [Mg2+].

2010/7/7 - ED Holmstrom, DJ Nesbitt

An approach for high spatiotemporal control of aqueous sample temperatures in confocal microscopy is reported. This technique exploits near-IR diode-laser illumination to locally heat picoliter volumes of water via the first-overtone excitation in the OH-stretch manifold.

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