Aptamer–ODN Chimeras: Enabling Cell-Specific ODN Targeting Therapy

Abstract

Oligonucleotides (ODNs) such as siRNA, saRNA, and miRNA regulate gene expression through a variety of molecular mechanisms and show unique potential in the treatment of genetic diseases and rare diseases, but their clinical application is still limited by the efficiency of the delivery system, especially the problem of the insufficient targeting of extrahepatic tissues. As homologous nucleic acid molecules, aptamers have become a key tool to improve the targeted delivery of ODNs. Aptamer–ODN chimeras can not only bind to multiple proteins on the cell surface with high specificity and selectivity, but they can also internalize into cells. Furthermore, they outperform traditional delivery systems in terms of cost-effectiveness and chemical modification flexibility. This review systematically summarizes the origin and progress of aptamer–ODN chimera therapy, discusses some innovative design strategies, and proposes views on the future direction of aptamer-ODN chimeras.

1. Introduction

Oligonucleotides (ODNs) such as small interfering RNA (siRNA), small activating RNA (saRNA), and microRNA (miRNA) are nucleic acid polymers that exhibit significant therapeutic potential for the treatment and management of various diseases. Their mechanisms of action primarily involve the precise regulation of gene expression through multiple molecular pathways, including but not limited to RNA interference (RNAi), RNase H-mediated cleavage, alternative splicing regulation, programmed gene editing, and gene activation [1]. They exhibit target specificity through two distinct interaction modalities: the majority act through canonical Watson–Crick base pairing with complementary nucleic acid targets to achieve sequence-specific recognition, while a minority, exemplified by aptamers and small guiding RNAs (sgRNAs), interact with proteins by forming three-dimensional secondary structures [1]. Compared to traditional drugs, ODN-based therapeutics have the potential to target previously undruggable pathogenic genes and patient-specific sequences associated with rare diseases [2]. As of March 2025, a total of 20 ODN-based therapeutic products have received regulatory approval from the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of a diverse range of diseases [3]. Despite the considerable progress in the clinical application of ODN-based therapeutics, the lack of effective delivery methods, particularly for targeted extrahepatic tissue distribution and tissue-specific cell surface receptor engagement, remains a critical translational barrier in the field. Numerous combinatorial strategies have been developed to enhance ODN delivery to target cells, including but not limited to lipid-based nanoparticles, peptide conjugates, and inorganic nanocarriers [4,5,6]. Furthermore, the conjugation of therapeutic ODN with nucleic acid aptamers has been extensively investigated as a promising approach to improve cell-specific targeting and intracellular delivery efficiency [7].

Since aptamers are also composed of nucleotides, they exhibit the same chemical and biological properties as ODNs such as a base pairing ability, chemical modification compatibility, etc. [8]. Compared to other ODNs, aptamers demonstrate a dual functional advantage: they can not only specifically bind to their targets but also be internalized into cells after binding, making them a powerful vehicle for the targeted delivery of ODNs [7]. Through molecular engineering techniques, aptamers have been extensively explored for programmable and facile coupling with diverse therapeutic ODNs, enabling their targeted delivery (Figure 1) [9]. Aptamer–ODN chimeras show superior advantages over other ODN delivery methods in terms of production cost, flexibility in chemical modification, and safety [10].

This review systematically summarizes the origin and progress of aptamer–ODN chimera therapy, discusses innovative design strategies, and proposes views on the future direction of aptamer-ODN chimeras.

2. Aptamers

2.1. Introduction and Application

Aptamers are synthetic, short (approximately 20–100 nucleotides in length), single-stranded DNA or RNA molecules that fold into defined three-dimensional structures, enabling them to bind target molecules with high specificity and affinity [11]. They are selected through an iterative in vitro process known as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The classical SELEX technique involves the following key steps: A highly diverse random ODN library containing approximately 10¹⁴ to 10¹⁶ unique nucleic acid molecules is constructed. The library is incubated with the target molecule, allowing specific nucleic acid sequences to bind to the target. Through methods such as elution and affinity purification, the ODNs that bind to the target are separated from the rest of the library and then amplified using PCR (for DNA libraries) or RT-PCR (for RNA libraries) to obtain enriched sequences. The amplified ODNs are re-incubated with the target molecule, and the binding, separation, and amplification steps are repeated. Typically, 6 to 20 rounds of selection are performed to progressively enrich the sequences with high affinity and specificity. The final enriched ODNs are sequenced, characterized, and cloned to identify the aptamers with the desired properties [12]. Leveraging their unique three-dimensional folding patterns and molecular recognition capabilities, aptamers can be systematically generated against a broad spectrum of analytes, ranging from simple metal ions and small organic molecules to complex biological entities, including proteins, viruses, bacteria, and even whole cells [13].

Aptamers are widely used in various fields of molecular biology, biotechnology, and biomedical sciences, particularly in gene therapy and targeted therapeutics [14,15,16,17]. Aptamer-based therapies primarily function through two mechanisms: (1) Direct target modulation: Aptamers act as antagonists or agonists to block or activate disease-related molecules. A prominent example is Pegaptanib, the first clinically approved aptamer drug, which selectively binds and neutralizes the vascular endothelial growth factor A165 (VEGF-A165), thereby suppressing pathological angiogenesis and vascular hyperpermeability in conditions like wet age-related macular degeneration [18]. (2) Therapeutic delivery: Aptamers serve as carriers to transport agents (e.g., siRNA, miRNA, chemotherapeutics, or toxins) to specific cells or tissues, enhancing local efficacy while minimizing systemic toxicity. Beyond these roles, aptamers can functionalize nanomaterials (e.g., nanoparticles, liposomes, polymeric micelles, or DNA nanostructures) to enable targeted drug delivery [19]. While existing reviews comprehensively cover aptamer-nanomaterial systems, this review focuses on aptamer–ODN chimeras, excluding detailed discussions of nanomaterial-based approaches [20,21,22,23,24].

2.2. The Internalizing Capacity of Aptamers

The internalization of aptamers is particularly important for in vivo applications in targeted delivery [25]. The vast majority of aptamers (such as the transferrin receptor (TfR) aptamer and protein tyrosine kinase 7 (PTK7) aptamer) enter cells through receptor-mediated endocytosis (RME). The specific mechanism of this process is as follows: The aptamer binds to specific receptors on the cell surface to form a complex. The complex then undergoes ligand-receptor clustering on the membrane surface, thereby triggering clathrin-mediated endocytosis (CME) or caveolae-mediated endocytosis (CvME) to internalize the complex into the cell. Notably, a small number of aptamers (e.g., nucleolin aptamers) can achieve receptor-independent internalization through macropinocytosis [25,26]. Afterwards, the aptamers either escape from the endosomes or are transported to lysosomes for degradation. Further studies have revealed that the cellular uptake pathway of aptamer-drug conjugates (ApDCs) is identical to that of aptamers. The aptamer directs ApDC to specific endocytic pathways, while the small-molecule drug does not interfere with their targeting or endocytosis. This confirms that the aptamer indeed plays a dominant role in the internalization of ApDCs [27].

There are two common ways to obtain cell-internalizing aptamers, one involves generating aptamers against predefined cell membrane protein targets; while the other, known as cell-internalization SELEX (also referred to as cell-SELEX or whole-cell SELEX or WC-SELEX), enables the generation of aptamers for target cells without the need to identify the target protein prior to the SELEX process [28,29]. WC-SELEX allows aptamers to be generated in their native environment even when structural information about the target is not yet known. In addition, by tuning the SELEX method, aptamers with unique intracellular behaviors can be developed [30]. Aptamers selected through both methods, targeting membrane proteins with internalizing properties, are being utilized to develop targeted delivery systems by conjugating them to therapeutics such as ODNs and toxic drugs.

2.3. Aptamers as Vehicles for Targeted Delivery of ODN

Targeted drug delivery necessitates precise therapeutic agent transport to specific sites/cells while minimizing off-target toxicity. Aptamers serve as ideal targeting ligands for this purpose. Compared with traditional targeting ligands such as antibodies, aptamers offer the following unique advantages: (1) a compact size that enables deeper tissue penetration; (2) exceptional thermal/chemical stability which supports long-term storage and functional recovery post-denaturation; (3) cell-free automated synthesis that ensures precision, reproducibility, and scalability; (4) flexible chemical modifications which enhance stability and enable conjugation with diverse materials; and (5) low immunogenicity/toxicity due to biocompatibility. In contrast, antibodies face the following limitations: temperature-sensitive inactivation, complex/costly live-cell production (risk of contamination), conformational disruption upon modification, poor tissue permeability due to a large size, and immunogenicity (e.g., ADA responses with murine/chimeric forms) [31,32,33,34]. With the above advantages, aptamers are increasingly used in targeted drug delivery.

Leveraging their unique advantages, aptamers can be programmably conjugated with diverse ODNs via molecular engineering, emerging as a core technology for ODN-target delivery. Compared with traditional ODN delivery systems (e.g., lipid nanoparticles or polymeric nanoparticles), aptamer–ODN chimeras exhibit the following features: (1) precise targeting: aptamers directly bind target cells via their intrinsic 3D structures, without requiring surface modifications like other systems; (2) high safety: Composed entirely of nucleic acids, aptamer–ODN chimeras show minimal immunogenicity, making them suitable for long-term or repeated administration, avoiding immune activation or the long-term accumulation risks associated with conventional carriers; (3) enhanced tissue penetration: their small size facilitates infiltration into dense tissues (e.g., solid tumors), while rapid renal clearance reduces systemic toxicity; and (4) scalable production: solid-phase synthesis ensures batch-to-batch consistency, with lower costs and complexity compared to nanocarrier fabrication. While aptamer–ODN chimeras also face limitations, such as susceptibility to nuclease degradation and difficulty in escaping from endosomes [35].

3. Aptamer–ODN Chimeras

Following the landmark breakthrough by Giangrande, PH’s team in designing the first aptamer-siRNA chimera [36], research on aptamer–ODN chimeras has expanded significantly. The types of ODNs delivered by aptamers have diversified to include siRNA, ASO, miRNA, and others. These ODNs can flexibly target the DNA or mRNA of key signaling pathway proteins (e.g., STAT3, Bcl-2), which are critical to disease-related processes such as signal transduction, cell proliferation, immune regulation, and apoptosis. Concurrently, the diversity of aptamer targets has rapidly increased, with common binding sites including disease-associated cell surface membrane proteins such as PSMA, HIV-1 gp120, EpCAM, CD4, HER2, EGFR, and nucleolin [37,38,39,40,41,42]. This broad target spectrum endows aptamer–ODN delivery systems with wide therapeutic applicability.

The combinatorial strategies of aptamer–ODN chimeras have evolved from simple covalent linkages to more sophisticated designs, including sticky bridge ligation, chemical crosslinker-mediated conjugation, and modular assembly systems [43]. Covalent linkage refers to the connection of two ODN chains using a phosphodiester bond, in which case the aptamer–ODN chimeras can be synthesized by solid-phase synthesis. Sticky bridge ligation utilizes designed complementary sequences (“sticky bridges”) to form hybrid structures that guide the ligation of two short nucleic acid strands. Chemical crosslinker-mediated conjugation employs bifunctional chemical reagents (e.g., glutaraldehyde, DSP) to directly form covalent bonds between molecules. This method enables the conjugation of nucleotides with other molecules, such as proteins. Modular assembly systems treat nucleic acid molecules (such as aptamers and ODNs) as independent functional modules, which can be combined in a controlled and flexible manner through predefined interfaces and connection strategies. This approach emphasizes module standardization and diversified connection strategies, enabling different functional components to be freely assembled like building blocks, thereby facilitating the rapid construction of chimeras tailored to specific functional requirements. Different connection strategies can be selected according to design requirements. These evolving conjugation strategies have significantly advanced the development of aptamer–ODN chimeras for therapeutic applications.

3.1. Aptamer-siRNA Chimeras

Small interfering RNA (siRNA) represents a class of short synthetic RNA duplexes, typically consisting of 21–23 nucleotide base pairs, with each strand terminating in characteristic 2-nucleotide 3′ overhangs. The mechanism of action involves the recruitment of siRNA duplexes into the RNA-induced silencing complex (RISC). An RISC is composed of multiple proteins and a small RNA molecule (e.g., siRNA, miRNA). One of the core components is the Argonaute protein, especially Ago2 (Argonaute 2), which is a key enzyme in RISC and has endonuclease activity that can catalyze the cutting of target mRNA. Within the RISC machinery, ATP-dependent strand separation occurs, resulting in the selective retention of the antisense (guide) strand within the activated RISC. The guide strand is complementary to the target mRNA, facilitating a sequence-specific recognition of complementary mRNA targets through Watson–Crick base pairing. Subsequent endonucleolytic cleavage, mediated by the PIWI domain of the Ago2 catalytic subunit, disrupts mRNA integrity and abrogates translational elongation [44,45,46,47]. siRNA exhibits exceptional therapeutic potential due to its high target gene specificity, relatively low immunogenicity, and straightforward design and validation processes [48]. Despite the therapeutic potential of siRNA, its clinical translation is hindered by delivery challenges, including rapid degradation in circulation and inefficient intracellular delivery. Consequently, designing targeted and stable delivery systems is essential. The covalent conjugation of aptamers with siRNA has emerged as a rational delivery paradigm. This approach offers advantages such as enhanced cellular uptake efficiency, improved cell-type specificity through aptamer-mediated targeting, and a reduced reliance on additional delivery vehicles, thereby improving the tolerability and safety profile of the delivery formulation [49,50].

Table 1. Overview of the research progress of aptamer-siRNA chimera therapy.

ODNs	Aptamer’s Name/Target	Target	Disease	Year	Ref.
siRNA	A9/PMSA	laminA/C	prostate cancer	2006	[51]
	A10/PMSA	PLK1 and BCL2	prostate cancer	2006	[36]
	A10-3.2/PSMA	PLK1	prostate cancer	2009	[52]
	PSMA aptamers/PSMA	Smg-1/Upf-2	colon cancer	2010	[53]
	PSMA aptamers/PSMA	Smg-1/Upf-2	prostate cancer	2013	[54]
	A10-3/PSMA	DNA-PK	prostate cancer	2015	[55]
	PSMA aptamers/PSMA	EGFR and survivin	prostate cancer	2016	[56]
	A10-3.2/PSMA	MDM2	prostate cancer	2022	[37]
	A10-3.2/PSMA	CAT-1	prostate cancer	2022	[57]
	4-1BB aptamer/4-1BB	mTORC1	antitumor immunity	2014	[58]
	4-1BB aptamer/4-1BB	CD25	antitumor immunity	2017	[59]
	gp120 aptamer/gp120 glycoprotein	HIV-1 gag p17	HIV	2008	[60]
	gp120 aptamer/gp120 glycoprotein	HIV-1 tat/rev	HIV	2009	[61]
	gp120 aptamer/gp120 glycoprotein	HIV-1 tat/rev	HIV	2011	[62]
	gp120 aptamer/gp120 glycoprotein	HIV-1 tat/rev	HIV	2011	[63]
	gp120 aptamer/gp120 glycoprotein	HIV-1 tat/rev	HIV	2011	[64]
	gp120 aptamer/gp120 glycoprotein	HIV-1 tat/rev	HIV	2013	[65]
	gp120 aptamer/gp120 glycoprotein	HIV-1 LTR	HIV	2018	[66]
	CCR5 aptamer/CCR5	TNPO3	HIV	2015	[67]
	CD4 aptamer/CD4	HIV genes and/or CCR5	HIV	2011	[68]
	CD4 aptamer/CD4	STAT5b	asthma	2012	[69]
	CD4 aptamer/CD4	HIV-PR	HIV	2012	[70]
	CD4 aptamer/CD4	CCR5	HIV	2013	[71]
	CD8AP17s/CD8	GNLY	immune diseases	2013	[72]
	BAFF-R aptamer/BAFF-R	STAT3	non-Hodgkin’s lymphoma	2013	[73]
	aptamer 32/U87-EGFRvIII	c-Met	gioma	2014	[74]
	aptNCL/nucleolin	SLUG and NRP1	lung cancer	2014	[75]
	AS1411/nucleolin	Bcl-2	breast cancer	2018	[76]
	AS1411/nucleolin	OPN	gioma	2022	[42]
	AS1411/nucleolin	SMG1	antitumor immunity	2022	[77]
	EpCAM aptamer/EpCAM	EpCAM	breast cancer/retinoblastoma	2015	[78]
	EpCAM aptamer/EpCAM	survivin	breast cancer	2015	[79]
	EpCAM aptamer/EpCAM	EpCAM	epithelial cancer	2015	[80]
	EpCAM aptamer/EpCAM	PLK1	breast cancer	2015	[81]
	EpCAM aptamer/EpCAM	D5D	colon cancer	2019	[82]
	EpCAM aptamer/EpCAM	PKCι	breast cancer	2020	[40]
	EpCAM aptamer/EpCAM	D5D	lung cancer	2020	[83]
	HER2 aptamer/HER2	Bcl-2	breast cancer	2012	[84]
	HER2, HER3 aptamer/HER2, HER3	EGFR	breast cancer	2018	[85]
	HER2 aptamer/HER2	EGFR	breast cancer	2018	[86]
	HER2 aptamer/HER2	XBP1	breast cancer	2020	[41]
	EGFR aptamer/EGFR	Bcl-2 and PKC-ι	breast cancer	2019	[87]
	EGFR aptamer/EGFR	XBP1	breast cancer	2021	[39]
	EGFR aptamer/EGFR	KRAS	non-small cell lung cancer	2023	[88]
	ErbB3 aptamer/ErbB3	survivin	breast cancer	2020	[89]
	sgc8c and sgc4f/PTK7	VEGF	leukemia	2016	[90]
	mucin-1 aptamers/mucin-1 (MUC1)	BCL2	multidrug-resistant breast cancer	2017	[91]
	VEGF aptamer/VEGF	notch 3	ovarian cancer	2017	[92]
	GBN/RGNNV coat protein (CP) protein	viral CP	viral nervous necrosis	2020	[93]
	LYGV1c/coat protein (CP) protein	SGIV MCP and VP19	grouper iridovirus infection	2021	[94]
	CTLA4 aptamer/CTLA4	STAT3	colorectal cancer	2014	[95]
	αvβ3 aptamer/αvβ3	EEF2	prostate cancer/cervical cancer/glioblastoma	2013	[96]
	Gint4.T/PDGFRβ	STAT3	glioblastoma	2018	[97]
	Gint4.T/PDGFRβ	STAT3	glioblastoma	2020	[98]
	Gint4.T/PDGFRβ	STAT3	non-small cell lung cancer	2023	[99]
	FB4/TfR	ICAM-1	neuroinflammatory disease	2014	[100]
	IGFIIR aptamer/IGFIIR	PCBP2	antifibrotic	2017	[101]
	monocytes/macrophages aptamer/monocytes/macrophages	ADK	immune diseases	2018	[102]
shRNA	CD40 aptamer/CD40	SMG1	B lymphoma and bone-marrow aplasia	2015	[103]
	CD40 aptamer/CD40	RORγt	immune diseases	2014	[104]
	CD30 aptamer/CD30	RORγt	autoimmune inflammatory diseases	2019	[105]
	S3R3/integrase	HIV-1 tat/rev	HIV	2018	[106]
	A10/PSMA	DNAPK	prostate cancer	2011	[107]

summarizes the progress in aptamer-siRNA chimera therapy.

The team led by Giangrande, PH, developed the first aptamer-siRNA chimera capable of delivering functional siRNA to specific cell types. This system combined a prostate-specific membrane antigen (PSMA) aptamer with siRNA targeting the mRNA of polo-like kinase 1 (Plk1) or B-cell lymphoma-2 (Bcl-2). PSMA is a transmembrane receptor that is highly expressed on prostate cancer cells and tumor-associated vascular endothelium, particularly in advanced metastatic castration-resistant prostate cancer (mCRPC), and its expression in other normal tissues is minimal. Therefore, it is an ideal therapeutic target in the treatment of prostate cancer [108]. Plk1 is a serine/threonine protein kinase that plays a central role in cell cycle regulation and is frequently overexpressed in tumor cells, making it a key therapeutic target in cancer [109]. Bcl-2, critical for regulating mitochondrial apoptosis pathways, inhibits programmed cell death. Its overexpression in certain cancers enables tumor cells to evade apoptosis and survive persistently, establishing it as a prominent target for anticancer therapies [110]. The PSMA aptamer-siRNA chimera bound with high affinity to PSMA through specific molecular recognition. Following RME, the chimera entered the target cells. Subsequently, the siRNA component underwent Dicer-mediated processing, ultimately leading to the degradation of Plk1- and Bcl-2-encoding mRNAs. By modulating both cell cycle progression and the apoptosis pathways, this chimera demonstrated potent therapeutic effects, including significant tumor growth suppression and observable phenotypic regression. The team further optimized the PSMA aptamer-siRNA chimera through chemical modifications, enhancing its silencing efficacy and specificity. In this design, the aptamer can be replaced with specific sequences targeting other tumor biomarkers (e.g., EGFR, HER2), while the siRNA can be customized to target the key mRNAs associated with various diseases, thereby providing a foundational framework for personalized treatment.

To enhance the target recognition capability of aptamers, multivalent aptamers were employed to construct aptamer-siRNA chimeras. For example, Barth, S et al. designed a bivalent aptamer-siRNA chimera by attaching the sense strand of the siRNA to the 3′ end of the first aptamer and the antisense strand to the 3′ end of the second aptamer. This configuration generated a precisely spaced 21-nucleotide double-stranded RNA (dsRNA) junction region (Figure 2a). Experimental validation revealed that the bivalent chimera exhibited both enhanced target affinity (while maintaining aptamer specificity) and a fourfold increase in internalization efficiency compared to the monovalent counterpart [111]. Subsequent studies utilizing similar bivalent architectures for siRNA delivery have reported comparable efficacy improvements [86,112].

Treatment approaches using chimeras to deliver two or more different siRNAs for combined therapy have also been developed. Liu, HY et al. developed a bivalent aptamer-dual siRNA chimera, which consists of a bivalent PSMA-specific aptamer and two distinct siRNA sequences targeting independent oncogenic pathways (EGFR and Survivin), along with a tetra-uracil (UUUU) spacer that serves as a functional linker to separate the siRNA duplexes (Figure 2b) [56]. Experimental validation demonstrated robust tumor-targeted delivery and the potent gene silencing efficacy of the EGFR- and survivin-targeting siRNAs, resulting in a significant reduction in tumor size and the inhibition of tumor-associated vasculature. The innovative bivalent aptamer-dual siRNA chimera strategy has opened new avenues for tumor-targeted therapy. Traditional single-targeted therapies may encounter challenges such as target escape and the development of drug resistance, whereas combined therapies can simultaneously target multiple carcinogenic pathways, thereby enhancing efficacy and reducing the risk of drug resistance.

Heterodimeric aptamer-siRNA chimeras were developed to address the problem of single-target resistance. The HER family comprises four functionally interdependent and compensatory members: the epidermal growth factor receptors (EGFR/HER1), HER2, HER3, and HER4 [113]. These receptors are closely related to tumor cell proliferation, invasion, and drug resistance and are overexpressed in tumor cells. Previous studies have demonstrated that antibody-based combinatorial targeting of EGFR, HER2, and HER3 achieves significantly higher efficacy than single-target approaches [114]. Inspired by this evidence, Liu, HY’s team, innovatively engineered a heterodimeric aptamer-siRNA chimera incorporating 2–4 unpaired nucleotide spacers between each aptamer and siRNA component (Figure 2c) [85]. This chimera integrates HER2- and HER3-specific aptamers with double-stranded EGFR-targeting siRNA, enabling each aptamer to perform dual roles: (1) acting as a receptor antagonist to inhibit ligand binding, and (2) serving as a cell-type-specific delivery vehicle for siRNA. Experimental validation revealed that the chimera simultaneously downregulated all three receptor expression levels, consequently inducing apoptosis. This innovative three-in-one design provides a novel therapeutic strategy to address the complexity of the HER family protein network, and the challenges associated with single-target therapy resistance. It highlights the significant potential of multi-targeted therapy in tumor immunotherapy, particularly for treating drug-resistant tumors and tumors activated by multi-receptors.

The three-way junction (3WJ) motif derived from the phi29 DNA packaging motor’s packaging RNA (pRNA) has been extensively used to link aptamers with diverse ODNs [57,82,83,88,115,116,117,118], demonstrating significant potential for developing RNA nanoparticles for disease treatment. The 3WJ architecture allows for the flexible modular loading of functional units, including the following: imaging modules (e.g., fluorophores or fluorescent aptamers), targeting modules (e.g., aptamers or chemical ligands), and therapeutic modules (e.g., siRNA, ASO, saRNA, miRNA, anti-miRNA, or chemotherapeutic agents). Each module operates independently while retaining structural and functional integrity (Figure 2d) [119,120]. These nanoparticles not only exhibit targeting and therapeutic effects but are also easy to image and track, providing a solid foundation for the widespread application of 3WJ nanoparticles in biomedical fields. Another chimeric RNA structure engineered from the phi29 DNA packaging motor also serves as a linkage bridge between aptamers and ODNs [63,69,100]. This structure can spontaneously form stable dimers through its unique staggered arrangement of left-hand and right-hand loops. The system consists of two core modules: the pRNA-aptamer module, which is responsible for the specific recognition and binding of cell surface receptors, and the pRNA-ODN module, which facilitates the efficient delivery of ODN (Figure 2e). This bifunctional chimeric system provides a novel technological platform for the development of cell type-specific ODN delivery vectors.

Short hairpin RNAs (shRNA) are synthetic RNA molecules composed of three components: a target-specific region, a spacer region, and a reverse complementary segment of the target sequence [121]. Compared to siRNA, shRNA contains an additional loop structure. After transcription, shRNAs are cleaved into small double-stranded RNA molecules by processing enzymes such as Dicer within the cell, converting them into functional siRNAs that enter the RNA interference (RNAi) pathway to target and degrade specific mRNAs [122]. Dicer is an important enzyme that cleaves long double-stranded RNA or precursor miRNA into small RNA fragments, initiating the gene silencing process. The combination of shRNA and aptamer follows a design concept similar to siRNA-aptamer chimeras. Specifically, these chimeras achieve targeted delivery by linking shRNA with specific receptor aptamer, allowing for the precise delivery of shRNA to particular cells or tissues. The aptamer component enhances the efficient entry of shRNA molecules into target cells by recognizing and binding to specific receptors on the cell surface, while the shRNA inhibits the expression of target mRNAs specifically through RNAi mechanisms within the cells [103,104,105].

Unlike siRNA, shRNA can be stably expressed via expression vectors. Therefore, by incorporating the aptamer sequence into the shRNA expression vector via a rational design, the stable expression of the aptamer-shRNA chimera can be achieved. Rossi, JJ’s team, inserted an anti-HIV integrase aptamer into the loop region of the Tat-Rev shRNA. HIV integrase is a key enzyme in the HIV replication cycle, responsible for integrating viral DNA into the host genome, thereby enabling long-term latency and continuous viral replication [123]. Tat and Rev are two key regulatory proteins in the HIV-1 lifecycle, playing central roles in the transcriptional activation of viral genes and RNA nuclear export, respectively. The design of this expression vector allows for the continuous production of large amounts of aptamer-shRNA chimeras through transcription (Figure 2f), thus achieving the long-term inhibition of HIV replication in cell culture systems [106]. The combination of shRNA and aptamers can be flexibly used to target the same gene (an mRNA and its corresponding protein), two genes (an mRNA and a different protein), or a protein and a non-coding RNA. Moreover, by using multiplexing vectors, multiple shRNA-aptamer chimeras can be expressed from a single transcript, enabling the simultaneous inhibition of multiple targets, which is of significant importance in combating the ever-evolving HIV.

3.2. Aptamer-ASO Chimeras

Therapeutic antisense oligonucleotides (ASOs) are synthetic, single-stranded DNA or RNA molecules, typically 18–30 nucleotides in length [124]. Their mechanisms of action vary depending on factors such as the target sequence, chemical composition, and nucleotide type. For example, ASOs can act through RNase H-mediated mRNA degradation, the spatial blockage of translation initiation, or the modulation of splicing patterns [125,126,127]. Depending on the mechanism, ASOs can target coding or non-coding RNAs, increase or decrease RNA or protein levels, as well as mask or disrupt miRNA and protein interactions, offering great flexibility in use [127,128]. ASO therapeutics offer several advantages, including a flexible design architecture that allows for sequence optimization, ease of chemical modification to enhance stability and specificity, scalable and reproducible synthesis protocols, and well-established quality control parameters. However, a major obstacle to clinical translation lies in developing effective delivery systems capable of overcoming biological barriers [129,130,131]. Aptamer–ASO chimeras offer a promising solution by facilitating targeted delivery, reducing the required ASO dosages and associated off-target effects, and improving safety [7].

Table 2. Overview of the research progress of aptamer-ASO chimera therapy.

ODNs	Aptamer’s Name/Target	Target	Disease	Year	Ref.
ASO	AS1411/nucleolin	luciferase	pancreatic cancer	2012	[132]
	TfR aptamer/TfR	caspase-3	ischemic stroke	2013	[133]
	S6 aptamer/A549	MMP-9	lung cancer	2015	[134]
	AS1411/nucleolin	galectin-1	breast cancer	2016	[135]
	Apt-2/LAMP1	MALAT1	lung cancer	2021	[28]
	AS1411/nucleolin	c-myc	lung cancer	2021	[136]
	PA9-1/PD-L1	PD-L1	antitumor immunity	2023	[137]
	SApt/SARS-CoV-2 spike protein	SARS-CoV-2 nucleocapsid	SARS-CoV-2	2023	[138]

summarizes the research progress in aptamer-ASO chimera therapy.

Aptamer-ASO chimeras exhibit excellent targeted delivery and therapeutic capabilities. Nucleolin is a ubiquitous, multifunctional protein predominantly located in the nucleolus, nucleoplasm, cytoplasm, and cell membrane. It is overexpressed in various cancers, and plays a multifaceted role in tumor initiation and progression [139]. AS1411 is a guanosine-rich ODN aptamer that binds to nucleolin and is subsequently internalized by tumor cells [140]. Kotula, JW et al. designed an aptamer-ASO chimera composed of an AS1411 aptamer targeting nucleolin and a splice-switching oligonucleotide (SSO) targeting luciferase. This chimera demonstrated an approximately 5-fold greater potency than the SSO alone in the test cells, highlighting the therapeutic value and nuclear delivery capacity of aptamer chimeras [132]. Similarly, Hong, SN et al. constructed an AS1411 aptamer-galectin-1 ASO chimera, which also demonstrated improved cellular uptake and selective entry into cells [135]. Tanaka, K et al. employed cell-internalization SELEX technology to screen and modify cell-internalizing aptamers for delivering ASOs into A549 cells, thereby accelerating the use of these aptamers as ODN delivery vehicles [28].

As mentioned above, aptamers can function as either agonists or antagonists in therapy, as well as drug delivery vehicles, enabling them to play a dual role in drug delivery [11]. Programmed death-ligand 1 (PD-L1) is an immune-regulatory protein with a complex subcellular distribution and multiple isoforms. Its receptor, PD-1, interacts with PD-L1 to form an immune checkpoint pathway that plays a critical role in tumor immune evasion. Tumor cells or tumor-associated immune cells frequently overexpress PD-L1, which binds to PD-1 on T cells, thereby inhibiting T cell function and evading immune clearance [141]. Current therapeutic strategies mainly target membrane-bound PD-L1 (mPD-L1), but new evidence suggests that intracellular and other forms of PD-L1 also influence anti-cancer immune responses [142]. Luo, FT et al. developed a novel antagonistic aptamer-ASO chimera with dual functionality: PD-L1-specific antagonism mediated by the aptamer domain, and targeted PD-L1 suppression via ASO-mediated gene silencing (Figure 3a) [137]. This bifunctional construct leverages the aptamer’s antagonistic capacity to disrupt PD-L1/PD-1 interactions, while concurrently facilitating the intracellular delivery of the conjugated ASO, enabling comprehensive PD-L1 suppression through both a surface receptor blockade and total protein downregulation. This strategy enhances immune activation compared to single-mechanism inhibitors and amplifies therapeutic outcomes through the synergistic effect of a checkpoint blockade and gene silencing, representing a promising and broadly applicable ODN-based immunotherapy strategy.

In addition to targeted delivery, a major challenge in ODN therapeutics is their inherent susceptibility to nuclease-mediated degradation. To address this limitation, Yang, G et al. pioneered the development of a circularized aptamer-ASO chimera designed for the dual suppression of SARS-CoV-2 viral proliferation and the associated inflammatory responses [138]. This innovative construct consists of three functionally integrated components: (1) A spike-targeting aptamer (SApt): a high-affinity ligand that specifically recognizes the trimeric spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It blocks viral entry by competitively inhibiting the spike protein–ACE2 receptor interaction and serves as a targeting moiety for delivering the chimera to infected cells. (2) A nucleocapsid-specific ASO (NASO): a therapeutic ODN engineered to silence the SARS-CoV-2 nucleocapsid (N) gene, thereby mitigating the N protein-mediated inflammatory cascades. (3) An optimized linker sequence: a structural element that preserves molecular flexibility and ensures the functional accessibility of the aptamer and ASO domains (Figure 3b). The circularized aptamer-ASO chimera significantly increases the stability of ODN in serum, establishing a novel strategy for ASO stabilization. This design overcomes the intrinsic limitations of linear architectures, and enables multifunctional integration within single-molecule entities, providing forward-looking strategies for molecular medicine.

3.3. Aptamer-miRNA Chimeras

As highly conserved endogenous regulatory molecules, microRNAs (miRNAs) are a class of non-coding RNAs (ncRNAs), typically 22 nucleotides in length. They mediate post-transcriptional gene silencing through the formation of incomplete base-pairing between their 5′-end seed region (nucleotides 2–8) and the 3′-untranslated region (3′-UTR) of target mRNAs [143]. This sequence-specific regulatory mechanism enables miRNAs to simultaneously regulate the expression networks of hundreds of target genes, playing a central role in biological processes such as cell cycle progression, proliferation, differentiation, metabolic homeostasis, and programmed cell death. Consequently, miRNAs are regarded as novel therapeutic targets with significant potential [144]. Given the abnormal expression profiles of miRNA in various pathological states, miRNA therapies can take the form of mimics, inhibitors, or combinations with other drugs. Mimic therapies involve introducing synthetic miRNAs to complement low-expressed functional miRNAs. These mimics re-suppress their target genes and subsequently normalize cellular processes to inhibit disease progression. In contrast, miRNA inhibitory therapies include the introduction of anti-miRNA ODNs (anti-miRs), miRNA masking, and miRNA sponges. Anti-miRNA ODNs (AMOs) have sequences complementary to the target miRNA, thus preventing the miRNA from binding to its mRNA target [145].

Achieving the efficient delivery of miRNA-based therapies remains a key bottleneck in clinical translation. Aptamers provide an innovative solution to overcome this limitation. The design methods for the aptamer-based delivery of miRNAs and anti-miRs involve ligating aptamers to miRNAs or anti-miRs using strategies such as sticky bridge technology or chemical crosslinking (Figure 3c). These aptamers can be rapidly internalized by cells expressing their specific targets, providing an effective tool for delivering miRNAs and anti-miRs [115,116,117,146,147,148,149,150,151,152,153,154,155,156,157,158,159] (

Table 3. Overview of the research progress of aptamer-miRNA chimera therapy.

ODNs	Aptamer’s Name/Target	Target	Disease	Year	Ref.
miRNA	CD133 aptamer/CD133	Anti-miR21	triple-negative breast cancer	2019	[117]
	EGFR aptamer/EGFR	Anti-miR21	breast cancer	2015	[116]
	EGFR aptamer/EGFR	Anti-miR21	breast cancer	2021	[154]
	A549 aptamer/A549	miR-301b-3p	lung adenocarcinoma	2023	[118]
	α9-nAChR aptamer/α9-nAChR	Anti-miR21	breast cancer	2023	[115]
	MUC1 aptamer/MUC1	PTEN	ovarian cancer	2013	[155]
	GL21.T/Axl	HMGA2	non-small cell lung cancer	2014	[153]
	GL21.T/Axl	miR-222	glioblastoma	2015	[147]
	GL21.T and Gint4.T/PDGFRβ and Axl	miR-137 and anti-miR-10b	glioblastoma	2016	[151]
	GL21.T/Axl	PED/PEA-15	non-small cell lung cancer	2016	[152]
	GL21.T/Axl	AXL	non-small cell lung cancer	2018	[150]
	GL21.T/Axl	Axl and the miR-137	non-small cell lung cancer	2019	[156]
	AS1411/nucleolin	c-raf-1	non-small cell lung cancer	2018	[157]
	AS1411/nucleolin	miRNA let-7d	gastric cancer	2019	[149]
	A10–3.2/PSMA	Bcl-2, cyclin D1, Wnt3a	prostate cancer	2011	[158]
	cKIT aptamer/cKIT	Ezh2 and Bak1	breast cancer	2023	[159]
	apt69.T/B cell maturation antigen	miR-137 and miR-122	multiple myeloma	2019	[146]

).

MiRNA degradation is also an effective strategy of miRNA therapy. RNA-inducible ribonuclease-targeted chimeras (RIBOTACs) are designed to achieve precise RNA degradation inspired by the concept of protein degradation-targeted chimeras (PROTACs) [160,161]. Their core molecular architecture consists of two functional domains: an RNA-binding domain, which achieves the specific recognition of target RNAs via ASOs or small molecule ligands; and an RNase L-recruiting domain, which recruits and activates ribonuclease to cleave the RNA, leading to its degradation [160]. RNase L recruiters can be either four 2′-5′-linked oligoadenylate units (4A), which are naturally present in cells, or small molecules obtained through DNA-encoded library screening. Building on this technology, Fang, Y et al. pioneered the aptamer-RIBOTAC (ARIBOTAC) platform by designing a 4A-ASO-AS chimera composed of three parts: (1) an intermediate ASO that exclusively recognizes the target miRNAs; (2) a 5′-end of the ASO coupled with the 4A structure that recruits and activates endogenous RNase L via monomer dimerization; and (3) a 3′-end AS1411 aptamer for targeting tumors and facilitating intracellular delivery. The 4A-ASO-AS chimera is specifically internalized into cancer cells overexpressing nucleolin via AS1411, followed by the recruitment and activation of RNase L by the 4A structure and miRNA recognition by the ASO. The formation of this ternary complex narrows the distance between the miRNA and RNase L, allowing the conserved motifs of the target miRNA to be localized in the catalytic center of RNase L. This mechanism enables site-specific cleavage and demonstrates a stronger antitumor effect than traditional miRNA inhibitors in murine models (Figure 3d) [162]. The modular design of ARIBOTAC technology offers a high degree of programmability: (1) different miRNAs (e.g., let-7 family or miR-155) can be targeted by replacing the ASO sequence; (2) the aptamer element can be substituted with other tumor-specific ligands such as EpCAM or PSMA; and (3) the entire structure, including the 2′–5′A activation module, can be optimized to enhance nuclease resistance. This flexible architecture provides a molecular basis for the development of a “universal” RNA degradation platform, which holds great translational value for the treatment of miRNA dysregulation-associated tumors and RNA virus infections.

3.4. Aptamer-saRNA Chimeras

Small activating RNAs (saRNAs) share high structural homology with siRNAs, consisting of 21 base pairs of double-stranded RNA with a characteristic 3′ overhang at the 3′ end. However, these two types of non-coding RNAs exhibit opposing effects on gene regulation [163]. Unlike siRNAs, which silence gene expression, saRNAs enhance the expression of target genes, leading to transcription levels that exceed basal expression. This unique gene-activating property makes saRNAs valuable in translational medicine, particularly in the field of therapeutic gene upregulation [164]. Current clinical studies on saRNAs primarily focus on the transcriptional activation of the CCAAT/Enhancer Binding Protein Alpha (CEBPA, or C/EBPα) gene for the treatment of leukemia and solid tumors [165]. CEBPA is a transcription factor involved in regulating various cellular processes, and its downregulation is strongly associated with the pathology of many malignant tumors. MiNA Therapeutics (a company) developed CEBPA-51 saRNA to specifically upregulate the transcription of the CEBPA gene and restore its expression [166]. This saRNA has demonstrated anti-tumor efficacy in different preclinical models using various delivery systems.

Table 4. Overview of the research progress of aptamer-saRNA/decoy chimera therapy.

ODNs	Aptamer’s Name/Target	Target	Disease	Year	Ref.
saRNA	P19/P1 aptamer/PDAC	C/EBPα	pancreatic cancer	2016	[167]
	TR14/hTfR	C/EBPα	prostate cancer	2019	[168]
	A10-3.2/PSMA	DPYSL3	prostate cancer	2016	[169]
	m12-3773/clusterin and 1-717/TMED6	XIAP	prostate cancer	2022	[170]
decoy	c2.min/hTfR	NF-κB	pancreatic cancer	2015	[171]
	GS-24/TfR	NF-κB	cerebrovascular inflammation	2015	[172]

summarizes the research progress in aptamer-saRNA/decoy chimera therapy.

Rossi, JJ et al. conjugated CEBPA-saRNA molecules with different aptamers to construct two composite delivery systems that specifically recognize pancreatic ductal adenocarcinoma (PDAC). They coupled CEBPA-saRNA with either a PDAC-specific aptamer or an anti-human transferrin receptor (hTfR) aptamer. The PDAC-specific aptamer was screened and optimized using whole-cell SELEX to ensure specific recognition of cell surface epitopes. hTfR is a type II transmembrane glycoprotein receptor that mediates the endocytosis and transport of trivalent ferric ions. Its expression can be upregulated nearly 100-fold in malignant, proliferating cell populations, making it an ideal molecular target for tumor-targeted therapy [173]. Studies have demonstrated that the targeted delivery of CEBPA-saRNA using either anti-hTfR or PDAC-specific aptamers leads to efficient endocytosed by pancreatic cells, resulting in a significant increase in CEBPA transcript and protein levels, as well as a notable tumor-suppressive effect [167,168,174].

3.5. Aptamer-Decoy Chimeras

Decoy technology includes two main approaches, decoy ODNs and decoy peptides [175]. Decoy ODNs are short, double-stranded molecules that mimic the DNA binding sites of the gene promoters and compete with native DNA sequences for transcription factor binding, thereby altering the expression of all downstream genes regulated by those transcription factors. This mechanism holds significant potential for regulating the transcription of disease-associated genes [176]. However, compared to ASO or siRNA methods, decoy ODNs are less specific in their mechanism of action. Their lack of tissue and cell specificity, along with susceptibility to degradation during endocytosis, limits their applications. Therefore, it is necessary to establish an effective delivery mechanism to transport decoys to the desired site of action [177]. In this context, aptamers can be used to facilitate the targeted delivery of decoys.

The double-stranded region in the middle of the aptamer–ODN chimera can be used to load with chemotherapeutic agents such as doxorubicin (Dox) to achieve synergistic therapy. Porciani, D et al. designed an aptamer-decoy chimera (aptacoy), consisting of an aptamer targeting the transferrin receptor (TfR) and a nuclear factor κB (NFκB) decoy. The two strands are assembled into an overall structure through complementary pairing at their ends, and Dox is loaded into the double-stranded DNA structure of aptacoy (Figure 4a) [171]. TfR is overexpressed in many solid tumors, while NF-κB is a ubiquitous transcription factor that suppresses Dox-induced apoptosis [178]. The experimental results showed that the aptamer motif facilitates the efficient and selective delivery of therapeutic molecules to tumor cells, while the combination of Dox and the NF-κB decoy enhances cellular sensitivity to DOX and amplifies its pro-apoptotic effects. The design of aptacoy aims to address the key challenges of poor selectivity and drug resistance associated with traditional chemotherapeutic drugs through the three-pronged strategy of “targeted delivery + drug loading + signaling pathway intervention”.

3.6. Aptamer-sgRNA Chimeras

CRISPR-Cas9 is a gene-editing technology capable of making precise cuts and modifications at specific locations in DNA molecules. As a representative of the third generation of gene editing technology, the CRISPR-Cas9 system has triggered a paradigm shift in the field of molecular genetics due to its programmability, efficiency, and precision [183]. The core components of this system include the Cas9 nuclease and single-guide RNA (sgRNA), which form an RNA-DNA heteroduplex with the target DNA via a spacer sequence at the 5′ end of the sgRNA. Cas9 functions as a molecular scissor, cutting the DNA at the target site, and resulting in double-stranded DNA breaks (DSBs). Subsequently, the cellular repair mechanism introduces modifications at the target site through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) [184,185]. In clinical applications, the safe and effective delivery of CRISPR/Cas9 remains a critical challenge. Delivery strategies can be categorized into three major types based on their molecular format: (1) a plasmid DNA (pDNA) vector system, which involves circular plasmids that can encode the Cas9 and sgRNA; (2) mRNA delivery systems that can express both Cas9 and sgRNA; and (3) the direct delivery of Cas9 and sgRNA as a ribonucleoprotein (RNP) complex. Each approach has its own advantages and disadvantages, as well as specific challenges that must be addressed [186].

Extracellular vesicles (EVs) are emerging as promising tools for delivering RNP complex. However, the lack of an efficient enrichment mechanism for loading RNPs into EVs limits their utility as delivery vehicles [187]. Previous studies have shown that the com (the aptamer named com)/ABP Com (the aptamer-binding protein named Com) pair can package Cas9 and adenine base editor RNPs into lentiviral capsids [188]. Based on this, Yao, XG et al. developed an aptamer-mediated method for RNP loading that enhances the incorporation of RNPs into EVs by leveraging specific interactions between com (inserted into the sgRNA) and Com (fused to CD63). In this technology system, the aptamer com element is assembled into a RNP complex with the Cas9 protein by inserting into the sgRNA backbone. As a signature membrane protein of exosomes, the tetraspanin CD63 has a topological structure with a transmembrane domain that penetrates the exosome membrane, with the N-terminus and C-terminus both located on the cytoplasmic side, and the hydrophilic ring structure extending into the exosome cavity [189]. To achieve precise positioning, the ABP Com was fused to the N-terminus, C-terminus, or both ends of CD63 to ensure that the Com functional domain was stably positioned at the cytoplasmic interface of the plasma membrane. To further improve the efficiency of exosome delivery, the research system co-expressed vesicular stomatitis virus G glycoprotein (VSV-G), which facilitates exosomal escape from endosomes in recipient cells via its membrane fusion capabilities. During exosome generation, the entire RNP complex is enriched in exosomes through interactions involving CD63-Com, com-Com, com-sgRNA, and Cas9. Notably, this enrichment mechanism has pathway diversity; in addition to the classical exosome pathway, RNPs can also be encapsulated in microvesicles formed by budding from the plasma membrane (Figure 4b) [179]. A quantitative analysis showed that this targeted enrichment system could increase gene editing activity by more than ten times compared with the control exosomes lacking an aptamer com or ABP Com. This innovative design not only provides an efficient solution for gene editing delivery, but also establishes a generalizable technical paradigm for engineering modifications in EVs.

In the coupling of aptamers and ODNs, the aptamers do not always function solely as targeting ligands [190]. Wu, HB et al. designed a DNA hydrogel composed of the CD123-targeting aptamer SS30 using rolled-loop amplification (RCA) methods, referred to as the SS30 polyaptamer hydrogel (SSFH). As a therapeutic aptamer, SS30 effectively inhibits the proliferation of acute myeloid leukemia (AML) cells by blocking the IL-3/CD123 signaling pathway, showing significant anti-tumor potential [191,192]. The research team designed two functionalized RCA templates: Template 1 contained the SS30 sequence and the sgRNA target sequence 1, while Template 2 incorporated the SS30 and the sgRNA target sequence 2 complementary to target sequence 1. After amplification, the complementary regions of the two templates enabled specific hybridization, forming a three-dimensional SSFH hydrogel. During the treatment implementation phase, when the SSFH and Cas9/sgRNA complex were co-injected into the target site, the Cas9 precisely cut the hydrogel skeleton by recognizing the specific site guided by the sgRNA, thereby continuously releasing the active SS30 aptamer in a controlled manner (Figure 4c). This design achieves the intelligent release of aptamer through a DNA hydrogel system regulated by CRISPR-Cas9. It not only provides a new strategy for the treatment of AML with high efficiency and low toxicity, but also redefines the role of aptamers and CRISPR tools in therapeutic delivery systems. The coupling of aptamers and sgRNAs also holds great potential in other applications [193,194,195] (

Table 5. Overview of the research progress of aptamer-CpG/sgRNA chimera therapy.

ODNs	Aptamer’s Name/Target	Target	Disease	Year	Ref.
cpg	IL-4Rα/IL-4Rα receptor	CpG-induced pathways	antitumor immunity	2017	[196]
	AS1411/nucleolin	CpG-induced pathways	antitumor immunity	2018	[181]
	AS1411/nucleolin	CpG-induced pathways	antitumor immunity	2024	[197]
	Aapt/ATP	CpG-induced pathways	antitumor immunity	2021	[198]
	ROX40 aptamer/ROX40	CpG-induced pathways	antitumor immunity	2025	[199]
	MUC1 aptamer/MUC1	CpG-induced pathways	antitumor immunity	2019	[200]
	PD-L1 aptamer/PD-L1	CpG-induced pathways	metastatic lung cancer	2023	[201]
	PD-L1 aptamer/PD-L1	CpG-induced pathways	antitumor immunity	2022	[202]
	PD-L1 aptamer/PD-L1	CpG-induced pathways	post-operative tumor recurrence	2023	[182]
others	com/Com	sgRNA	duchenne muscular dystrophy	2021	[179]
	PD-1 aptamer/PD-1	sgRNA	antitumor immunity	2019	[190]
	SS30/CD123	sgRNA	acute myeloid leukemia	2019	[180]
	NCL-APT/nucleolin	survivin	retinoblastoma	2021	[203]
	MUC1 aptamer/MUC1	Egr-1	breast cancer	2020	[204]
	Sgc8/PTK7	MET	lung cancer	2020	[205]
	CD44-EpCAM aptamer/CD44 and EpCAM	CD44 and EpCAM	ovarian cancer	2017	[206]
	TfR-aptamer/TfR	Tau	tauopathy	2020	[207]

).

3.7. Aptamer-CpG Chimeras

CpG ODNs are synthetic single-stranded DNA molecules characterized by a specific sequence (CpG motif), which consists of tandemly linked unmethylated cytosine (C) and guanine (G) connected by phosphodiester bonds. By mimicking the naturally occurring CpG motifs in pathogen DNA, these molecules can specifically activate Toll-like receptor 9 (TLR9) in immune cells, triggering MyD88-dependent signaling pathways that induce the secretion of a variety of cytokines, thereby enhancing the innate immune response and promoting the activation of adaptive immunity. Due to this mechanism, CpG ODNs have been widely used in tumor immunotherapy, anti-infective vaccine adjuvant and allergic disease treatment [208,209]. Despite preclinical studies confirming their significant immune-activating potential, the clinical application of CpG ODNs is hindered by several limitations, including difficult cellular uptake, poor stability, rapid degradation, low targeting efficiency, a short half-life, and insufficient tissue penetration. Therefore, effective delivery strategies must be explored to maximize their therapeutic efficacy and accelerate their clinical translation [210,211]. One promising approach involves the combination of CpG ODNs with aptamers.

Precision delivery and intelligent controlled release are important research directions for improving drug efficacy. Han, Y et al. designed an immunoglobulin nanomedicine named the imHDL/Apt-CpG-Dox composite system. In this design, the AS1411 aptamer was fused with a CpG motif to create an Apt-CpG sequence. This Apt-CpG was then conjugated to the synthetic phospholipid DSPE(1,2-distearoyl-sn-glycero-3-phospho-ethanolamine) to form an Apt-CpG-DSPE molecule, which was subsequently incorporated into the lipid monolayers of high-density lipoprotein (HDL) to construct imHDL/Apt-CpG nanoparticles. HDL is commonly used for drug delivery due to its customizable structure and high biocompatibility. Finally, Dox was loaded into the double-stranded ODN region between the CG base pairs, yielding the complete imHDL/Apt-CpG-Dox composite system (Figure 4d) [181]. Apolipoprotein AI (Apo-AI), the main component of HDL cholesterol, is the ligand for the scavenger receptor class B type I (SRBI), which is expressed at higher levels in many tumor cells and serves as a potential biomarker for tumor diagnosis and prognosis [212]. Once Apo-AI is recognized by the SR-BI receptors at the tumor site, the HDL nanostructures trigger extracellular dissociation, releasing Apt-CpG-Dox into the tumor cell mesenchyme. The AS1411 aptamer in Apt-CpG-Dox further binds to the membrane nucleolin and translocates to the nucleus, where Dox is released and exerts its therapeutic effects. Meanwhile, the CpG motifs induce antigen recognition, pro-inflammatory cytokine secretion, and host anti-tumor immunity. Ultimately, the synergistic action of these components results in an enhanced anti-tumor effect. This design builds an efficient and safe multifunctional nanodrug platform by integrating the targeting capabilities of natural HDL carriers, the membrane and nuclear localization capabilities of AS1411, the immunostimulatory effect of CpG, and the chemotherapeutic effect of Dox. Its core advantage lies in maximizing therapeutic efficacy while minimizing side effects through precise delivery and intelligent controlled release, providing an innovative strategy for tumor treatment.

Wang, DY et al. developed an intelligent DNA hydrogel system that integrates tumor recurrence monitoring and photodynamic immune regulation. The system is based on two functionalized circular DNA templates: one encodes the PD-L1 aptamer and the CpG sequence, while the other encodes the tumor recurrence sensor binding domain. These templates are co-assembled with the photosensitizer Ce6-cDNA through a one-step rolling circle amplification (RCA) process. Further, a circular ATP sensor (cAS) is incorporated into the hydrogel network through a sequence complementation strategy to construct a CPDH-Ce6@cAS composite system (Figure 4e) [182]. In the clinical translation design, the hydrogel can be implanted in situ in the surgical area after tumor resection. The PD-L1 aptamer selectively captures early recurrent tumor cells, and the resulting cell aggregation effect significantly increases the local ATP concentration. The cAS component responds to the ATP gradient, activating a fluorescence signal to enable real-time monitoring of tumor recurrence. Upon detection of a positive signal, near-infrared (NIR) laser irradiation activates the Ce6-mediated photodynamic effect. The generated reactive oxygen species (ROS) not only directly kill tumor cells but also trigger the disassembly of the hydrogel network, thereby accurately releasing the PD-L1 aptamer and CpG sequence. The released PD-L1 aptamer blocks the PD-1/PD-L1 immune checkpoint, while the CpG motif stimulates the dendritic cell-T cell immune cascade reaction, ultimately achieving synergistic immunotherapy. This design integrates diagnosis, treatment, and immune regulation functions into a single platform through precise programming at the molecular level, realizing the closed-loop management of “monitoring–warning–treatment” for postoperative recurrence, providing an innovative strategy for the postoperative treatment of tumors, and demonstrating the application potential of precision medicine in the field of integrated molecular diagnosis and treatment.

4. Conclusions

Since the discovery of aptamers in the 1990s, their applications in the biomedical field have gradually expanded, especially in targeted drug delivery systems [213]. With the development of ODN-based therapies, aptamers have been extensively explored for binding to various types of ODNs (such as siRNA, miRNA, ASO, etc.) to form aptamer–ODN chimeras. These chimeras not only facilitate targeted delivery, but also enable the release of ODNs following cellular internalization, thereby achieving therapeutic purposes such as gene silencing [214]. At present, aptamer–ODN chimeras have shown promise in various research fields, especially in cancer, viral infections, and genetic diseases [35,215]. Despite their broad potential, the widespread application of aptamer–ODN chimeras in the biomedical field still faces several challenges. First, the challenge of ODN release: even after successful internalization, the chimeras must escape from endosomes/lysosomes to release the ODNs, and cellular uptake efficiency remains difficult to control. Second, the challenge of nucleic acid degradation: both aptamers and ODNs are nucleic acid molecules and are susceptible to degradation by nucleases. Moreover, extensive preclinical studies and clinical trials are necessary to ensure the safety and efficacy of these chimeras in humans. Finally, as a novel therapeutic strategy, aptamer–ODN chimeras may encounter complex regulatory approval processes, and determining how to meet regulatory requirements represents an additional hurdle.

In order to expand its application in biomedicine and related fields, the research on aptamer–ODN chimeras can focus on the following directions: (1) The optimization of design strategies: a. The enhancement of molecular stability: Aptamers and ODNs are easily degraded by nucleases in vivo and typically exhibit short half-lives. To improve their stability, chemical modifications are necessary. They may include thiophosphate bonds (PS backbone), 2′-O-methylation (2′-OMe), and locked nucleic acids (LNA) to enhance resistance to nucleases such as RNase H and serum nucleases. The aptamer backbone and ODN bases can be optimized separately to avoid mutual interference [32]. Moreover, circular aptamer–ODN structures can significantly improve stability by eliminating nuclease recognition sites, thus enhancing resistance to degradation [138]. b. The design of precision targeting systems: creating precise targeting systems that allow aptamers to recognize multiple distinct cell surface receptors or multiple copies of the same receptor, thereby improving specificity and targeting accuracy [216]. c. The design of intelligent controlled release systems: controlled-release mechanisms, such as redox-driven, enzyme-driven, light-driven, or heat-driven mechanisms, can be employed to enable stimulus-responsive ODN releases under specific circumstances, thereby reducing off-target effects [217]. d. Integration with immunotherapy or chemotherapy: Combining aptamer–ODN chimeras with immunotherapy or chemotherapy could produce synergistic therapeutic effects, ultimately enhancing overall efficacy. Aptamer–ODN chimeras can also be combined with emerging technologies such as CRISPR gene editing technology or nanomedicine to further improve the targeting, controllability, and the efficacy of disease treatment. (2) The optimization of SELEX technologies: With the widespread application of aptamers as targeting ligands, traditional WC-SELEX has been unable to meet actual application needs due to its inherent limitations such as long experimental cycle and complex operation process. To overcome these technical bottlenecks, it is necessary to develop new and efficient aptamer screening systems. For instance, Fluorescence-activated cell sorting-assisted WC-SELEX (FACS-WC-SELEX) combines FACS with WC-SELEX. In this method, the random library is labeled with fluorescein. After incubation and washing, cells are analyzed using a flow cytometer. Upon laser excitation, aptamer-bound cells emit fluorescent signals, which are quantified by the FACS system to isolate positive cells. This method significantly shortens screening time, improves aptamer enrichment, and enhances specificity. Microfluidic chip system-assisted WC-SELEX (MC-WC-SELEX) integrates micro-fluidic technology with WC-SELEX to automate the on-chip SELEX process. Compared to conventional WC-SELEX, MC-WC-SELEX is faster, highly automated, and can complete 15 rounds of selection in just 3 days, while requiring fewer cells and reagents. The 3D culture-assisted WC-SELEX (3D-WC-SELEX) employs target cells cultured in a 3D environment that mimics physiological conditions, thereby enhancing the relevance and selectivity of aptamer screening. Nanomaterial-assisted WC-SELEX (NA-WC-SELEX) incorporates suitable nanomaterials into the selection process to shorten the screening time and improve aptamer performance [30]. Continuous innovation in technologies such as high-throughput screening, AI algorithms, and single-round SELEX technologies is essential to identify more suitable aptamers that meet application needs [31,218,219,220]. (3) The optimization of large-scale production: Long-chain chimeras (>80 nucleotides) are prone to generating truncated products, posing significant challenges for large-scale manufacturing. The production of high-quality, cGMP-grade chimeras remains a major hurdle in therapeutic development. Solving these problems may be done from multiple aspects, such as optimizing the synthesis process, optimizing the purification method, and exploring the enzymatic in vitro synthesis platform [214]. (4) An in-depth study of the intracellular fate of chimeras: For almost all aptamer-ODN chimeras delivery, their fate within the cell, especially the transitions required to cross lipid biolayers, such as to escape from endosomal vesicles, remains elusive [221]. Moreover, there is a lack of quantifications of escape efficiency. Therefore, it is necessary to gain a deeper understanding of the intracellular fate of the chimeras, further study their internalization mechanism, and establish a standardized evaluation system to quantify the differences in endosomal escape rates of different designs.

In summary, we have reviewed recent progress in aptamer–ODN chimeras. As a novel targeted delivery system, aptamer–ODN chimeras offer notable advantages such as precise targeting, a strong cellular internalization ability, and cost-effectiveness, showing broad application potential across multiple therapeutic fields. However, several challenges remain to be addressed in order to promote the development of aptamer–ODN chimeras and expand their applications in biomedicine and related areas, as discussed above. With ongoing advancements in nucleic acid chemistry, delivery system engineering, SELEX technologies, and other relevant fields, the clinical potential of aptamer–ODN chimeras is expected to be fully realized.