Mass Spectrometry for Assessing Protein–Nucleic Acid Interactions

Stability-Based Proteomics for Investigation of Structured RNA–Protein Interactions

“…While the overlap was limited due to fundamental differences in the methodologies, the overlapping hits include literature-confirmed, direct binders of TH such as ILF3 and the HNRNP proteins. While affinity-based pull-downs and stability methods are both useful for identifying strong, direct RNA–protein interactions on the proteome scale, only stability-based methods can provide information on indirect interactions like disrupted protein–protein interactions and conformation-related events that can occur within the proteome upon RNA treatment. , At least some of the hits unique to the stability experiments are hypothesized to interact with the RNA in indirect ways such as in the case of Sam68, which was found to be destabilized by TH addition in both TPP and SPROX (Table S3). This TH -induced destabilization is likely due to the disruption of Sam68 interactions with other proteins.…”

“…For example, covalently modified in vitro transcribed RNAs are costly to synthesize and may adopt non-native conformations due to the modifications. Further, the results of pull-down experiments are typically skewed toward the detection of high affinity, direct interactions and frequently miss weaker, more transient interactions. , Pull-down approaches also miss potential secondary interactions that could be impacted by these RNA–protein interactions (e.g., protein–protein interactions that may be disrupted by RNA binding). It is essential to develop new tools to overcome these limitations and allow native RNA structural dynamics and protein interactions to be interrogated.…”

“…RNA–protein interactions are critical to the biological function of RNA transcripts, and these interactions have long been investigated in vitro by using affinity-based capture methods. − These methods have included adding biotinylated in vitro transcribed RNAs to cell lysates that are then exposed to streptavidin-labeled beads to pull down bound proteins, , using dT-oligo beads to pull-down UV cross-linked proteins to poly­(A)-containing RNA sequences, and using biotinylated tiling oligos to target and pull down full RNA transcripts along with their associated proteins or nucleic acids. , Multiple studies have employed these techniques coupled with mass spectrometry to identify protein interactions for RNA sequences − as well as RNA structures. ,− Unfortunately, pull-down approaches have significant experimental drawbacks. For example, covalently modified in vitro transcribed RNAs are costly to synthesize and may adopt non-native conformations due to the modifications.…”

“…Reported here are the results of an innovative adaptation and evaluation of the SPROX and TPP approaches for studying RNA–protein interactions. Unlike conventional affinity pull-down approaches, , these techniques do not require modification of the RNA molecule, and they are amenable to the analysis of RNA–protein binding interactions with a wide range of affinities. As part of this work, the SPROX and TPP methods are used to study three different RNAs with varying structure (primary, secondary, and tertiary) and their influence on the folding stability of proteins in LNCaP nuclear lysate.…”

Evaluation of aconitine cardiotoxicity with a heart-on-a-particle prepared by a microfluidic device

“…2 Currently, most in vitro heart models revolve around tissue engineering constructs, such as 3D methacrylated gelatin scaffolds patterned via microscale continuous optical printing to mimic aligned three-dimensional cardiac microstructures, 3 as well as the utilization of stretchable sensors for monitoring the sequential contraction of cardiomyocytes. 4 Although these models successfully replicate the internal cardiac environment, their operational procedures are comparatively burdensome and limited in terms of portability and scalability.…”