A sensitive RNA chaperone assay using induced RNA annealing by duplex specific nuclease for amplification
Graphical abstract
Introduction
The RNA chaperone Hfq is a small, abundant, ubiquitous protein, which is conserved in a wide range of bacterial phyla and plays an important role in posttranscriptional gene regulation by interacting with several small RNAs by base pairing and is required for their function [[1], [2], [3], [4], [5], [6]]. Hfq forms a ring-shaped homo-hexamer that specifically binds sRNAs and mRNAs, which affects the stability of several mRNAs and targets them for degradation by increasing polyadenylation, interfering with ribosome binding and with translation [[7], [8], [9], [10]]. The association of mRNAs and siRNAs depends on their sequences and secondary structures, and is typically inefficient at the low mRNA concentrations in the living cell [[11], [12], [13]]. Hfq also acts as an RNA chaperone by appearing to bind preferentially to unstructured A/U-rich sequences, frequently close to more structured regions of the RNA, and increases the rate of base pairing with mRNA targets, and stabilized siRNA-mRNA complexes [[14], [15], [16], [17], [18]]. It belongs to the Hfq that is structurally and functionally related to the distant archaeal and eukaryotic homologues. Hfq binds to A/U-rich sequences encoded sRNAs as well as their target mRNAs, thus facilitating base pairing between the two strands with special sequence complementarity [[14], [15], [16], [17], [18]]. Hence, Hfq binding duplex formation may subsequently regulate gene expression at the level of RNA stability or translation.
Woodson group reports that Hfq forms a transient ternary complex with two RNA strands, increasing helix initiation 103 to 104 times above the uncatalyzed rate [7]. Based on this, they successfully developed a light triggered RNA annealing method by an RNA chaperon, Hfq. Although RNA annealing methods have been reported, more sensitive assay strategies to functional these RNA chaperons with RNA annealing are still necessary.
This assay employed duplex specific nuclease (DSN) enzyme for the amplification reaction. DSN is practically passive toward single-stranded DNA or RNA, or double-stranded RNA. However, it displays a strong preference for cleaving double-stranded DNA (more than 10 base pairs) or DNA with a high preference in DNA-RNA hybrid heteroduplex and leaves the original RNA intact so that it can bind to another DNA. Thus, a DNA sequence with signal indicator that is complementary to RNA sequences could be used as a specific RNA biosensor [19]. RNA-related research with elevated sensitivity assay could be achieved by introduction the DSN to the assay system [[20], [21], [22], [23], [24], [25], [26]].
In addition, molecular beacons (MBs) are single stranded oligonucleotide probes that possess a stem-and-loop structure [[27], [28], [29], [30], [31]]. The loop portion of the molecule can report the presence of a specific complementary nucleic acid. The base pairs at the two ends of the MB are complementary to each other, forming the stem. When the probe encounters a target DNA or RNA molecule, it forms a hybrid that is more stable than the stem, and its rigidity and length preclude the simultaneous existence of the stem hybrid [[31], [32], [33], [34], [35], [36], [37]]. Thus, the MB undergoes a spontaneous conformational reorganization that forces the stem apart. Therefore, the MBs may lead to signal change by combined the MB with other signal transducer when hybridized to their target molecules.
In this manuscript, we report an ultrasensitive Hfq-aided RNA annealing assay strategy that uses DSN amplification method. In this strategy, the Hfq-induced reaction and DSN digestion can convert the RNA annealing to the Cy3 fluorophores fluorescence intensity with high sensitivity. More importantly, our method is suitable for the assay Hfq-induced RNA annealing in real samples.
Section snippets
Reagents
Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and Diethyl pyrocarbonate (DEPC) were purchased from Sigma-Aldrich Inc. (St. Louis, Missouri, USA). The strand sequences were obtained from Genscript Biotech. Co., Ltd. (Nanjing, China) with the sequences as shown in Table 1. Duplex-specific nuclease (DSN) was purchased from Newbornco Co., Ltd (Shenzhen, China). Malachite Green (MB) was obtained from J&K Scientific Ltd. (Shanghai, China). Fluorescence was measured by RF-5301PC
The working principle
The working principle of the RNA chaperone induced RNA annealing by DSN based amplification method is illustrated in Scheme 1A. In this strategy, two molecular beacons (MB1 and MB2) were ingeniously designed. RNA1 consists of two fragment parts: a part of A12 for Hfq recognition and another part for annealing with complementary MB1. MB1 consists of two fragments: one for annealing with RNA1 and another for hybridization with MB2's loop part. In the initial state, the MB1 exists in the state of
Conclusions
In summary, we have developed a simple and ultrasensitive method for the real-time assay of RNA annealing reaction by coupling the DSN-aided signal amplification strategy with the Hfq triggered RNA annealing reaction. By employing a MB probe which modified 2-OMe-RNA at its stem part, we transform the RNA annealing reaction to the real-time fluorescence intensity of Cy3. Rather, the DSN-aided amplification reaction is a sensitivity approach to assay RNA annealing with a κrea of 0.16 s−1,
Acknowledgment
This work was supported by grants from the National Natural Science Foundation (21705061) and the Major Project of Wuxi Municipal Health Bureau (ZS201401, Z201508).
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