Circular Single-Stranded DNA Molecule Can Simultaneously Silence Multiple MicroRNAs

Professor Dongsheng Liu of Tsinghua University, Professor Ziyang Hao of Capital Medical University, and Researcher Yuanchen Dong of the Institute of Chemistry, Chinese Academy of Sciences, have collaborated to develop a circular single-stranded DNA molecule capable of simultaneously silencing multiple miRNAs.

This molecule can be used for multi-gene synergistic tumor therapy. Based on the KIMU principle, the circular single-stranded DNA molecule, an anti- miRNA oligonucleotide (circAMO), can be synthesized with high selectivity and high yield by adjusting the length of the DNA clamp. The unique covalently closed circular structure endows circAMO with high biostability, allowing long-term intracellular gene regulation without any chemical modifications. By designing multiple miRNA binding sites in circAM, one circAMO can simultaneously inhibit multiple oncogenic miRNAs and upregulate the levels of downstream mRNAs, ultimately inhibiting tumor cell proliferation, migration, and invasion, as well as increasing apoptosis. This strategy provides a new research tool and platform for multi-target gene therapy. The article was published as an open access research article in CCS Chemistry, the flagship journal of the Chinese Chemical Society.

Background Information:

MicroRNAs (miRNAs), as endogenous non-coding RNAs that regulate gene expression, are closely related to diseases such as cancer when dysregulation occurs. The 2024 Nobel Prize in Physiology or Medicine awarded to this field also confirms the potential of miRNA therapy. Using anti-miRNA oligonucleotides (AMOs) to target and downregulate overexpressed oncogenic miRNAs in tumors has shown good efficacy in the treatment of various solid tumors. However, AMOs without chemical modifications are easily degraded by nucleases and have poor stability. While chemical modification can improve stability, it poses biosafety risks and is easily cleared by the kidneys. Circular RNAs (circRNAs), although resistant to nucleases and capable of inhibiting miRNAs at multiple sites, are cumbersome to synthesize and require excessive use. Small circular DNA-based AMOs, while highly efficient, have low binding affinity. Therefore, there is an urgent need to develop AMOs that combine high stability, high affinity, and the ability to simultaneously inhibit multiple oncogenic miRNAs.

Highlights of This Article:

1. Selective synthesis of single-molecule circular single-stranded DNA through rigid regulation
Based on the kinetic interlocking mechanism previously proposed by our research group, the authors developed a one-pot method for the efficient synthesis of circular single-stranded DNA (cssDNA). The synthesis involves a two-step ligation reaction using T4 DNA ligase and CircLigase, followed by exonuclease III digestion and ultrafiltration purification. The size of the cssDNA can be precisely controlled by adjusting the length of the clamping DNA, enabling controllable synthesis. During the circLigase II-catalyzed cyclization process, Mn²⁺ can increase the yield of circAMO from approximately 20% to approximately 50%, and exonuclease III digestion removes linear byproducts generated during the two-step ligation process. Combined with rolling circle amplification (RCA) experiments and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), the synthesis of 132 nt circAMO was successfully verified (Figure 1).

2. Excellent structural and functional characteristics
circAMO is designed with four miRNA binding sites (MBS) targeting miR-21, miR-221, miR-155, and miR-10b, respectively, with 10-nucleotide spacers between each MBS. This reduces mutual interference during miRNA binding and alleviates structural tension in DNA/RNA hybridization. In vitro experiments show that circAMO can completely degrade these four oncogenic miRNAs, individually or simultaneously, within 10 minutes via an RNase H-mediated cleavage mechanism, achieving simultaneous inhibition of multiple targets (Figure 2).

The covalently closed topology of circAMO makes it highly resistant to exonucleases. It maintains its structural integrity for 24 hours under the action of exonuclease I and exonuclease V, while linear AMO (linAMO) degrades rapidly during this period. Under serum-containing cell culture conditions, circAMO retains its integrity for 36 hours, while linAMO degrades completely within 12 hours. Furthermore, circAMO exhibits higher intracellular stability; after transfection into MCF-7 cells, its fluorescence signal decays slowly, achieving a more durable miRNA inhibitory effect (Figure 3).

3. Highly efficient gene regulation and anti-tumor effects
In MCF-7 cells, circAMO can inhibit target miRNAs in a concentration-dependent manner (Figure 4). At each tested concentration, its inhibitory effect on miRNAs is better than that of linAMO. It can significantly upregulate the expression of downstream tumor suppressor mRNAs (PTEN, HIPK2, SOCS1, HOXD10), and the regulatory effect is stronger and more lasting. It can still maintain a certain regulatory effect after 72 hours, while the regulatory effect of linAMO disappears quickly (Figure 5).

Anti-tumor experiments showed that circAMO inhibited the proliferation of MCF-7 cells by 61% five days after transfection, which was 1.8 times that of linAMO (34%). Wound healing and Matrigel Transwell experiments showed that circAMO reduced cell migration by 58% and inhibited invasion by 56%. p-nitroaniline (pNA) hydrolysis experiments showed that the pNA accumulation caused by caspase-3/7 activation induced by circAMO was 1.8 times that of linAMO, and it could effectively induce tumor cell apoptosis (Figure 6).

Summary and Outlook:

In summary, this study successfully developed a circAMO platform based on circular single-stranded DNA, enabling the one-pot controlled synthesis of 132 nt circAMO. This circAMO exhibits high nuclease resistance and can simultaneously and irreversibly degrade four oncogenic miRNAs-miR-21, miR-221, miR-155, and miR-10b-through an RNase H-mediated cleavage mechanism, significantly upregulating the expression of downstream tumor suppressor mRNAs. In the MCF-7 breast cancer cell model, it demonstrated superior and more durable miRNA inhibitory effects and antitumor activity compared to linAMO, including inhibiting cell proliferation, migration, and invasion, and inducing apoptosis. This platform requires no chemical modification, solving the stability problem of traditional nucleic acid therapies and providing an innovative paradigm for multi-target oligonucleotide therapy. In the future, we need to optimize the purification process of circAMO to meet the material requirements of preclinical studies and ensure batch consistency, carry out functionalization studies such as targeted delivery, verify its in vivo pharmacokinetics, pharmacodynamics and long-term safety through animal models, and expand its application in other cancers and miRNA dysregulation-related diseases to provide support for clinical translation.