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Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction.

ABSTRACT: Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens are essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-O-methoxyethyl (2'-O-MOE) gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including phosphorothioate linkage, 2'-O-methyl, 2'-O-MOE, and locked nucleic acid, as well as siRNAs. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.

SUBMITTER: Shin M

PROVIDER: S-EPMC8817697 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction.

Shin Minwook M Meda Krishnamurthy Pranathi P Devi Gitali G Watts Jonathan K JK

Nucleic acid therapeutics 20211217 1

Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens are essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-<i>O</i>-methoxyethyl (2'-<i>O</i>- ...[more]

PMID: 34928745

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Project description:BackgroundNumerous models for use in interpreting quantitative PCR (qPCR) data are present in recent literature. The most commonly used models assume the amplification in qPCR is exponential and fit an exponential model with a constant rate of increase to a select part of the curve. Kinetic theory may be used to model the annealing phase and does not assume constant efficiency of amplification. Mechanistic models describing the annealing phase with kinetic theory offer the most potential for accurate interpretation of qPCR data. Even so, they have not been thoroughly investigated and are rarely used for interpretation of qPCR data. New results for kinetic modeling of qPCR are presented.ResultsTwo models are presented in which the efficiency of amplification is based on equilibrium solutions for the annealing phase of the qPCR process. Model 1 assumes annealing of complementary targets strands and annealing of target and primers are both reversible reactions and reach a dynamic equilibrium. Model 2 assumes all annealing reactions are nonreversible and equilibrium is static. Both models include the effect of primer concentration during the annealing phase. Analytic formulae are given for the equilibrium values of all single and double stranded molecules at the end of the annealing step. The equilibrium values are then used in a stepwise method to describe the whole qPCR process. Rate constants of kinetic models are the same for solutions that are identical except for possibly having different initial target concentrations. Analysis of qPCR curves from such solutions are thus analyzed by simultaneous non-linear curve fitting with the same rate constant values applying to all curves and each curve having a unique value for initial target concentration. The models were fit to two data sets for which the true initial target concentrations are known. Both models give better fit to observed qPCR data than other kinetic models present in the literature. They also give better estimates of initial target concentration. Model 1 was found to be slightly more robust than model 2 giving better estimates of initial target concentration when estimation of parameters was done for qPCR curves with very different initial target concentration. Both models may be used to estimate the initial absolute concentration of target sequence when a standard curve is not available.ConclusionsIt is argued that the kinetic approach to modeling and interpreting quantitative PCR data has the potential to give more precise estimates of the true initial target concentrations than other methods currently used for analysis of qPCR data. The two models presented here give a unified model of the qPCR process in that they explain the shape of the qPCR curve for a wide variety of initial target concentrations.

Dataset Information

Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction.

Publications

Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction.

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