ssDNA–dsRNAs are cleaved at the next to its chimera-junction point by an unknown RNase activity

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Abstract

We found that there is an unknown aspect in serum RNases that cleaves ssDNA–dsRNA and ssRNA–dsRNA. In the first step, RNase cleaves the phosphodiester linkage between the first and second RNA, where the first one is connected to the single stranded RNA or DNA. In the second step, the ssRNA overhang attached siRNA is cleaved. When the 2′ hydroxyl of the first RNA was replaced with methoxy, the cleavage did not occur. This RNase activity can be considered related to defense system against exogenous genetic materials.

Highlights

► ssDNA–dsRNA chimeras are cleaved in a position-specific manner in serum. ► The cleavage site is at the phosphodiester linkage between the first and second RNA. ► 2′ methoxylation of the first RNA prevents the cleavage. ► This activity of RNase may be related to siRNA defense system.

Introduction

RNA interference (RNAi) is an RNA-dependent gene-silencing process that occurs in cytosol. RNAi is initiated by the cleavage of long double-stranded RNAs (dsRNAs) into short dsRNAs by an enzyme called Dicer [1], [2]. The short dsRNAs, called small interfering RNAs (siRNAs) [1], [3], are usually composed of 20–26 nucleotides with an overhang at the 3′ end. When exogenous dsRNAs enter the cytosol, RNAi plays an important role in the immune response [4]. For endogenous dsRNAs such as miRNAs, RNAi is related to gene silencing or downregulation [5]. The application of RNAi in biotechnology began with the groundbreaking discovery that artificially designed short dsRNAs (∼21 nucleotides) can silence the expressions of certain endogenous genes homologous to the dsRNAs in mammalian cell lines [6]. This siRNA technology facilitated enormous progress in the study of gene functions. Although there is risk of activating the interferon response, many researchers are developing the therapeutic applications of siRNAs; some of them have reached clinical trials [7]. The development of a safe delivery method is critical for the clinical application of RNAi, because naked siRNAs are easily excreted via renal clearance and degraded in blood stream. Various delivery vehicles have been proposed in the last decades, including viral vectors, and cationic lipids and polymeric materials as non-viral vectors [8], [9]. During our study of such vehicles [10], [11], we found a strange phenomenon in which the junction point of single-stranded (ss) DNA–dsRNA was selectively cleaved in serum. This study describes this rather strange phenomenon and attempts to estimate its biological significance.

Section snippets

Chimera composed of ssDNA and dsRNA

All oligonucleotides were synthesized by Hokkaido System Science Co., Ltd. (Hokkaido, Japan) and purified with high-performance liquid chromatography. All sequences used in this study are described in Supplementary Table 1.

Stability of chimeras to serum

After the chimeras were incubated in 10% human serum (HS), fetal bovine serum (FBS) or mouse serum (MS) at 20 μg/ml for 1 h at 37 °C, the they (50 ng) were analyzed by a 12% polyacrylamide gel (PAGE) for 1 h at 100 V. After the gel was stained with SYBR Gold (Invitrogen, Carlsbad,

Cleavage of chimera in serum

We have been studying a series of ssDNA–dsRNA chimeras comprising oligo-deoxyadenine (dA40 and dA10) and one of siRNA sequences, denoted as siTNF [12] hereafter, in which the oligo-deoxyadenine is attached to the 5′ end of the sense strand of the siTNF sequence (Supplementary Table 1). Fig. 1A shows the extents of chimera degradation in FBS and HS. The chimera was cleaved into two fragments; its cleavage was suppressed by an RNase inhibitor RNaseOut™ (Invitrogen). Comparing the dA40 and siTNF

Discussion

The above results demonstrate that RNases have an unknown activity in HS and FBS, showing sequence- and position-selective cleavage of the chimera; this activity is shared with bovine RNase A and S. However, it is unlikely that naked endogenous nucleotides are involved in any biological reaction in serum. By eliminating such impossibilities, we can speculate that this RNase activity may be related to a host defense mechanism. RNases are secreted from organs including the pancreas and kidneys

Acknowledgments

We would like to gratefully thank Ms. Motoko Tanaka for her help. This work is financially supported by the JST CREST program.

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