Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures

Abstract

Molecular analysis of RNA through hybridization with sequence-specific probes is challenging due to the intrinsic ability of RNA molecules to form stable secondary and tertiary structures. To overcome the energy barrier toward the probe-RNA complex formation, the probes are made of artificial nucleotides, which are more expensive than their natural counterparts and may still be inefficient. Here, we propose the use of a multicomponent probe based on an RNA-cleaving deoxyribozyme for the analysis of highly structured RNA targets. Efficient interrogation of two native RNA from Saccharomyces cerevisiae-a transfer RNA (tRNA) and 18S ribosomal RNA (rRNA)-was achieved at ambient temperature. We achieved detection limits of tRNA down to ∼0.3 nM, which is two orders of magnitude lower than that previously reported for molecular beacon probes. Importantly, no probe annealing to the target was required, with the hybridization assay performed at 37°C. Excess of nonspecific targets did not compromise the performance of the probe, and high interrogation efficiency was maintained by the probes even in complex matrices, such as cell lysate. A linear dynamic range of 0.3-150 nM tRNA was demonstrated. The probe can be adapted for differentiation of a single mismatch in the tRNA-probe complex. Therefore, this study opens a venue toward highly selective, sensitive, robust, and inexpensive assays for the interrogation of biological RNA.

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“…This perfect selectivity is not surprising considering significant difference in the genomes of the alternative viruses and the ability of BiDz to differentiate single nucleotide substitutions in broad temperature ranges. [15] We then investigated the ability of 4DNM to accurately recognize the presence of CoV2 in human clinical samples. RNA samples were isolated from 14 patients according to the standard procedure for RNA preparation for RT-qPCR.…”

“…Another advantage is its high selectivity towards single nucleotide substitutions (SNS). [12,15] This is achieved by shortening Arm 2 complementary to the SNS site (Figure 1A). Short arm binds only the fully complementary, but not single base mismatched sequences.…”

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“…These LOD values, while unable to compete with the LODs of PCR-based assays ( Braun et al, 2021 ), are in the same range as the LOD reported for the molecular beacon probe (MBP) ( Kolpashchikov 2012 ). Unlike MBP, which cannot efficiently interrogate a structured nucleic acid target ( Nguyen et al, 2011 ; Reed et al, 2020 ), the split junction probe used in the aptamer-expressing assay can tolerate stable secondary/tertiary structures a natural RNA target generally acquires. Here, this advantageous property of the split probes was used to target native 16S rRNA sequences of bacterial species.…”

“…In this case, the affinity of the 3WJ probe to the target was maintained by the length of the TP strand. Previously, we have shown that even if one component of the split probe is shortened to allow for discrimination of point mutations, the length of the target-recognizing fragment of another probe’s component controls the overall affinity to keep the signal high ( Gerasimova et al, 2015 ; Reed et al, 2020 ). This illustrates the advantage of split hybridization probes over monolith ones—the ability to cope with the affinity-specificity dilemma that may compromise performance of monolith probes ( Demidov and Frank-Kamenetskii, 2004 ).…”

“…An alternative approach to NAATs is to amplify a signal rather than the nucleic acid target, for example, with the help of enzymes ( Gerasimova and Kolpashchikov 2014 ). Some of the signal-amplification strategies are paired with junction probes (also known as multicomponent, or binary, probes) ( Wang and Tao, 2020 ), which offer advantages of target differentiation down to a point-mutation resolution ( Kolpashchikov 2010 ), and interrogation of highly structured nucleic acids, such as rRNA or tRNA ( Reed et al, 2020 ). One type of the junction probes is based on the formation of the three-way junction (3WJ) structure, which is made of three duplex domains forming a junction point ( Wu et al, 2004 ).…”