LC–TOF–MS methods to quantify siRNAs and major metabolite in plasma, urine and tissues

Bioanalysis ◽  
2019 ◽  
Vol 11 (21) ◽  
pp. 1983-1992 ◽  
Author(s):  
Lakshmi Ramanathan ◽  
Helen Shen
Keyword(s):  
High Resolution ◽  
Small Molecules ◽  
Antisense Strand ◽  
Bioanalytical Method Validation ◽  
S1 Nuclease ◽  
Molecular Weights ◽  
Sense Strand ◽  
Tissue Lysate ◽  
Probe Hybridization ◽  
High Resolution Mass Spectrometer

There are a few different bioanalytical approaches that have been used for the quantification of siRNA in biological matrices, such as S1 nuclease protection ‘cutting ELISA’, fluorescent probe hybridization HPLC, HPLC UV, LC–MS/high-resolution accurate-mass (HRAM) and LC–MS/MS. We have developed and validated plasma assays for several oligonucleotides such as GalNAc-conjugated siRNA, using uHPLC and high-resolution mass spectrometer by TOF detection. Although the molecular weights are in the range of 7000–9000, we were able to meet the same assay acceptance criteria as for the small molecules based on regulatory bioanalytical method validation guidance. The antisense strand and the sense strand can both be monitored. The method was also used in the tissue lysate matrices without a full validation.

High-resolution nonlinear raman spectroscopy of small molecules

1986 ◽  
Vol 141 ◽  
pp. 195-202 ◽  
Author(s):  
H.W. Schrötter ◽  
H. Berger ◽  
B. Lavorel
Keyword(s):  
Raman Spectroscopy ◽  
High Resolution ◽  
Small Molecules

scholarly journals Stochastic steps in secondary active sugar transport

2016 ◽  
Vol 113 (27) ◽  
pp. E3960-E3966 ◽  
Author(s):  
Joshua L. Adelman ◽  
Chiara Ghezzi ◽  
Paola Bisignano ◽  
Donald D. F. Loo ◽  
Seungho Choe ◽  
...  
Keyword(s):  
High Resolution ◽  
Small Molecules ◽  
Sugar Transport ◽  
Ion Binding ◽  
Sugar Transporter ◽  
Human Homolog ◽  
Domains Of Life ◽  
Insight Into ◽  
Stochastic Functional ◽  
Unique Capability

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

Quantitative Structure Activity Relationship (QSAR) study predicts small molecule binding to RNA structure

2021 ◽  
Author(s):  
Zhengguo Cai ◽  
Martina Zafferani ◽  
Olanrewaju Akande ◽  
Amanda Hargrove
Keyword(s):  
High Resolution ◽  
Small Molecules ◽  
Small Molecule ◽  
Rna Structure ◽  
Quantitative Structure ◽  
Rna Structures ◽  
Qsar Study ◽  
Structure Activity ◽  
Qsar Models ◽  
Hiv 1

The diversity of RNA structural elements and their documented role in human diseases make RNA an attractive therapeutic target. However, progress in drug discovery and development has been hindered by challenges in the determination of high-resolution RNA structures and a limited understanding of the parameters that drive RNA recognition by small molecules, including a lack of validated quantitative structure-activity relationships (QSAR). Herein, we developed QSAR models that quantitatively predict both thermodynamic and kinetic-based binding parameters of small molecules and the HIV-1 TAR model RNA system. A set of small molecules bearing diverse scaffolds was screened against the HIV-1-TAR construct using surface plasmon resonance, which provided the binding kinetics and affinities. The data was then analyzed using multiple linear regression (MLR) combined with feature selection to afford robust models for binding of diverse RNA-targeted scaffolds. The predictivity of the model was validated on untested small molecules. The QSAR models presented herein represent the first application of validated and predictive 2D-QSAR using multiple scaffolds against an RNA target. We expect the workflow to be generally applicable to other RNA structures, ultimately providing essential insight into the small molecule descriptors that drive selective binding interactions and, consequently, providing a platform that can exponentially increase the efficiency of ligand design and optimization without the need for high-resolution RNA structures.

High-resolution mass spectrometer for protein chemistry

Nature ◽  
1994 ◽  
Vol 370 (6488) ◽  
pp. 393-395 ◽  
Author(s):  
Yunzhi Li ◽  
Richard L. Hunter ◽  
Robert T. Mclver
Keyword(s):  
High Resolution ◽  
Mass Spectrometer ◽  
Protein Chemistry ◽  
High Resolution Mass ◽  
High Resolution Mass Spectrometer ◽  
Resolution Mass
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