Elsevier

Analytica Chimica Acta

Volume 1033, 29 November 2018, Pages 199-204
Analytica Chimica Acta

A sensitive RNA chaperone assay using induced RNA annealing by duplex specific nuclease for amplification

https://doi.org/10.1016/j.aca.2018.05.073Get rights and content

Highlights

  • A new sensing platform for Hfq induced RNA Annealing is reported.

  • DSN amplification strategy is used for the sensitivity assay.

  • This sensing platform employed 2-OMe-RNA modified molecular beacon to prevent DSN cleavage.

  • Hfq induced RNA annealing rate of κrea = 0.16 s−1 was observed.

Abstract

The hybridization of two complementary RNAs in single cells depends on their complementary sequences and secondary structures, and is usual inefficient at the low concentrations. The bacterial RNA chaperone Hfq increases the rate of base pairing hybridization of mRNA, and stabilizes sRNA-mRNA duplexes. However, The RNA chaperone Hfq accelerates the RNA annealing between two complementary pair RNAs with a still unknown mechanism. So the sensitivity assay of Hfq induced RNA annealing is very important. By using a 2-OMe-RNA modified molecular beacon as a reporter, which can be specificity cleavage by DSN, we observed the amplification reaction kineticsrea) is 0.16 s−1. Our results showed that the Hfq hexamer directly induced the RNA annealing, and DSN aided the ultra-sensitivity assay reaction with 0.18 fM Hfq/RNA1/MB1.

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).

References (39)

  • T. Soper et al.

    Positive regulation by small RNAs and the role of Hfq

    Proc. Natl. Acad. Sci. Unit. States Am.

    (2010)
  • M. Beich-Frandsen et al.

    Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

    Nucleic Acids Res.

    (2011)
  • A. Santiago-Frangos et al.

    Acidic C-terminal domains autoregulate the RNA chaperone Hfq

    eLife

    (2017)
  • S. Panja et al.

    Light-triggered RNA annealing by an RNA chaperone

    Angew. Chem. Int. Ed.

    (2015)
  • A. Santiago-Frangos et al.

    C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

    Proc. Natl. Acad. Sci. Unit. States Am.

    (2016)
  • M. Doetsch et al.

    Study of E. coli Hfq's RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents

    Nucleic Acids Res.

    (2013)
  • C.M. Courtney et al.

    Sequence-specific peptide nucleic acid-based antisense inhibitors of TEM-1 β-lactamase and mechanism of adaptive resistance

    ACS Infect. Dis.

    (2015)
  • Y. Cao et al.

    Crispri–srna: transcriptional–translational regulation of extracellular electron transfer in shewanella oneidensis

    ACS Synth. Biol.

    (2017)
  • B. Tjaden et al.

    Target prediction for small, noncoding RNAs in bacteria

    Nucleic Acids Res.

    (2006)
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