Antisense oligonucleotides (ASOs) are a treatment modality for genetic diseases. ASOs bind to ribonucleic acid (RNA) with high specificity, with downstream effects that eventually reduce, restore, or modify protein expression through distinct mechanisms. Conjugating ASOs to targeting ligands has emerged as a promising strategy to improve tissue-specific delivery. Here, we develop streamlined assays that assess the binding kinetics of both the ASO and the targeting ligand using biolayer interferometry (BLI). The utility of this biomolecular sandwich binding assay is demonstrated with phosphorodiamidate morpholino oligomers (PMOs) and peptidenucleic acids (PNAs) conjugated to a peptide or protein ligand. We show that this assay can be used to detect intracellular uptake and predict in vitro efficacy. We believe that the methods developed here can accelerate the development of next-generation ASO therapeutics.

Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathies. Conventional methods used to identify Hb variants in clinical laboratories can narrow down the range of candidates for a Hb variant sample but are unable to pinpoint the exact Hb variant. In this study, next-generation protein sequencing (NGPS), a semiconductor chip-based single-molecule protein sequencing (SMPS) technology, was explored as a novel method to identify Hb variants. Two heterozygous Hb variant samples underwent NGPS analysis. Proteotypic peptides corresponding to Hb variants were successfully detected, enabling the identification of the samples as Hb Handsworth (Hb α-Handsworth subunit G18R) and Hb G-Accra (Hb β-G-Accra subunit D73N). The NGPS method has been demonstrated as a potential tool to identify Hb variants. Although there are still limitations to overcome for the wide adoption of NGPS, this exploration supports the potential use of NGPS and other SMPS technologies in clinical applications.

Proteins are a class of macromolecules with essential roles in processes and structures associated with life. Protein sequencing technologies are therefore fundamental for understanding cell metabolic pathways, disease mechanisms, and how pathogenic agents and toxins function. Emerging next generation protein sequencing (NGPS) technologies promise a dramatic improvement of proteomics methods enabling identification of pathogens and toxins with unparalleled sensitivity and precision. The Quantum-Si (QSi) Platinum Sequencer is a novel single molecule protein sequencing technology capable of single amino acid resolution. In this work, we conducted significant optimization of the QSi protein library preparation protocol, reducing sample preparation time from 32 to 10 hours without sacrificing substantial sequencing quality, allowing for a sample-to-answer timeline in less than 24 hours. The modified protocol was applied for analyzing a set of proteins including sixteen single-domain antibodies with diverse sequences and a nontoxic derivative of staphylococcal enterotoxin B. We were further able to determine the library dilution threshold: losing the ability to sequence beyond 100x dilution. Finally, we were able to successfully obtain protein sequences within a crude lysate background, demonstrating the effectiveness of sequencing within complex protein mixtures. Improvements in sequencing chemistry and data processing may soon lessen or eliminate the dependence on reference sequences: a current obstacle for efficiently characterizing unknown proteins. By further condensing and optimizing library preparation, this technique presents a potential application for proteomics that require rapid characterization of highly complex biological systems, significantly improving protein-based diagnostic technologies.

The vast complexity of the proteome currently overwhelms any single analytical technology in capturing the full spectrum of proteoform diversity. In this study, we evaluated the complementarity of two cutting-edge proteomic technologies—single-molecule protein sequencing and individual ion mass spectrometry—for analyzing recombinant human IL-6 (rhIL-6) at the amino acid, peptide, and intact proteoform levels. For single-molecule protein sequencing, we employ the recently released Platinum® instrument.

Protein variants of the same gene–proteoforms–can have high molecular similarity yet exhibit different biological functions. Thus, identifying unique peptides that unambiguously map to proteoforms can provide crucial biological insights.

Next-generation protein sequencing (NGPS) is a single-molecule approach for characterizing protein variants, offering detailed insight into proteoforms and amino acid substitutions not easily discerned by mass spectrometry.