Sources of mismeasurement of RNA knockdown by DNAzymes and XNAzymes

IF 3.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Maria J. Donde, Alicia Montulet and Alexander I. Taylor
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Abstract

RNA-cleaving oligonucleotide catalysts composed of DNA and/or nucleic acid analogues (DNAzymes, modified DNAzymes and XNAzymes) are promising agents for specific knockdown of disease-associated RNAs. However, we and others have identified discrepancies between their apparent activity in vitro versus when transfected into cells. Here, using examples of catalysts targeting the codon 12 region of KRAS RNA – an unmodified DNAzyme based on the classic “10–23” motif, a modified DNAzyme (“10–23_v46”) or an XNAzyme (“FR6_1_KRas12B”) – we examine confounding effects including unintended activity during standard RNA work-up steps, leading to mismeasurement of knockdown. We find that catalysts are not irreversibly denatured by typical cell lysis reagents, nor fully degraded by typical DNase treatments, exacerbated by nuclease resistant modification chemistries. In standard RT-qPCR workflows, DNAzymes and XNAzymes were found to be capable of cleaving their target RNAs during (1) DNase treatment and (2) reverse transcription (RT) reactions, in both instances with enhanced rates compared with under quasi-physiological conditions, producing cleavage-dependent false positives. Furthermore, catalysts were found to site-specifically inhibit cDNA synthesis (i.e. producing cleavage-independent false positives) and in the case of DNAzymes also had the capacity to act as primers during RT, leading to an enhancement of target site cDNA as judged by digital PCR, producing (cleavage-independent) false negatives. These effects could be broadly mitigated by purification to remove catalysts at the point of RNA extraction, under denaturing conditions. We recommend that studies of oligo catalysts in cells must include a 0 h timepoint after catalyst delivery or transfection to assess the collective impact of these mismeasurements on a case by case basis.

Abstract Image

DNAzymes和XNAzymes对RNA敲低错误测量的来源。
由DNA和/或核酸类似物(DNAzymes,修饰的DNAzymes和XNAzymes)组成的rna切割寡核苷酸催化剂是一种有前景的特异性敲除疾病相关rna的药物。然而,我们和其他人已经确定了它们在体外与转染到细胞中时的明显活性之间的差异。在这里,使用针对KRAS RNA密码子12区域的催化剂的例子-基于经典“10-23”基元的未修饰DNAzyme,修饰DNAzyme(“10-23_v46”)或XNAzyme(“FR6_1_KRas12B”)-我们检查了混杂效应,包括在标准RNA工作步骤中的意外活性,导致敲除的错误测量。我们发现,催化剂不会被典型的细胞裂解试剂不可逆地变性,也不会被典型的dna酶处理完全降解,更不会被耐核酸酶修饰化学物质加剧。在标准的RT- qpcr工作流程中,DNAzymes和XNAzymes被发现能够在(1)DNase处理和(2)逆转录(RT)反应中切割它们的靶rna,在这两种情况下,与准生理条件下相比,切割率更高,产生切割依赖的假阳性。此外,催化剂被发现可以位点特异性地抑制cDNA合成(即产生与切割无关的假阳性),并且在DNAzymes的情况下,在RT过程中也具有作为引物的能力,导致通过数字PCR判断的靶位点cDNA增强,产生(与切割无关的)假阴性。在变性条件下,这些影响可以通过在RNA提取点的纯化去除催化剂来广泛减轻。我们建议对细胞中低聚催化剂的研究必须包括催化剂递送或转染后的0小时时间点,以逐个评估这些错误测量的集体影响。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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