一、Introduction

Aptamers are single‑stranded nucleic‑acid molecules selected through in‑vitro SELEX. Unique three‑dimensional folded structures enable specific target recognition. Conventional mono‑target aptamers only bind one single molecule. They cannot meet research demands for multi‑target coordinated intervention. Bispecific aptamers carry two independent recognition modules. They bind two different targets at the same time. The two targets can be two separate receptors on one cell surface, or molecules sitting on two different cell types. This property creates molecular‑bridging effects ^[1].

Two major development routes exist for bispecific aptamers. The first route obtains two independent mono‑target aptamers by separate SELEX rounds, then joins them with linker segments for modular assembly. The second route applies Toggle‑SELEX. It alternates two target substances during screening to isolate naturally occurring dual‑target‑binding sequences.

Bispecific aptamers inherit favourable traits of nucleic‑acid molecules: small size, easy chemical modification and low immunogenicity. Yet assembly may bring conformational interference and reduced binding affinity. Supported by mature SELEX workflows, KMD‑Bioscience completes mono‑target aptamer screening, bispecific aptamer molecular construction and multi‑dimensional activity validation. It delivers one‑stop support for early‑stage bispecific aptamer research.

二、Technical Principles and Experimental Workflows

The core principle of bispecific aptamers: two functional aptamer units that each recognize one target get connected by nucleic‑acid linkers. Each unit folds into its own independent three‑dimensional conformation. They bind their corresponding targets separately and achieve dual‑target recognition. For Toggle‑SELEX workflows, nucleic‑acid libraries get exposed alternately to two different targets. Sequences capable of binding both targets get enriched to yield native bispecific aptamers ^[2,3].

Two mainstream experimental pipelines are available: modular assembly route and Toggle‑SELEX direct‑screening route.

Route One: Modular Assembly (most widely adopted) 

①Mono‑target screening

Run magnetic‑bead SELEX, Cell‑SELEX or CE‑SELEX against target A and target B separately. Isolate high‑activity aptamers for both targets.

②Molecular construction

Design nucleic‑acid linkers of varying length and flexibility. Connect the two aptamer fragments in tandem and build multiple candidate bispecific aptamer variants.

③Conformation and binding validation

Test whether assembled aptamers retain binding capacity for both targets. Rule out steric hindrance brought by linkers.

④Optimization and screening

Truncate and optimize linker length and sequences. Pick bispecific aptamers with best overall performance.

⑤Functional assessment

Carry out SPR measurement, flow‑cytometry tests and cell‑based functional assays to evaluate biological activity.

Route Two: Direct Screening via Toggle‑SELEX

①Incubate random nucleic‑acid libraries alternately with target A and target B. Swap targets in every SELEX round.

②Complete binding, separation, elution and amplification cycles in each round. Gradually enrich sequences that recognize both targets.

③Mine candidates by NGS sequencing. Verify whether candidates possess genuine dual‑target‑binding performance.

Toggle‑SELEX comes with high technical difficulty and low enrichment efficiency. Most bispecific aptamers in current research adopt the modular assembly approach.

三、Technical Advantages and Existing Challenges

1.Technical advantages

①Bispecific aptamers support coordinated dual‑target recognition. They create cell‑cell bridges. Immune cells get recruited toward tumour cells for immune redirection. They act as valuable tool molecules for tumour‑immunology studies [4].

②Assembly builds on well‑validated mono‑target aptamers from mature SELEX. Development timelines stay predictable, and molecular modification via chemical routes proves convenient.

③Compared with bispecific antibodies, bispecific aptamers have smaller molecular weight. Synthesis costs remain low, and batch‑to‑batch consistency performs well.

④They can block two separate signalling pathways at once. Applied in multi‑target molecular detection, they improve specificity of detection systems.

2.Major technical challenges

①After two aptamer segments get joined, linkers may introduce steric hindrance. Two aptamer modules interfere with each other’s folding. Binding affinity drops for one or even both target partners. This counts as the most frequent problem during bispecific aptamer construction.

②Toggle‑SELEX direct screening faces high barriers. Naturally occurring sequences capable of binding two targets are extremely rare inside libraries. SELEX enrichment works poorly.

③Nucleases degrade nucleic‑acid molecules in‑vivo, and renal clearance shortens circulation half‑life. Additional chemical modification becomes necessary.

④Dual‑target activity validation requires complex assay set‑ups. Researchers need to test binding for both targets instead of relying on single‑target read‑outs.

四、Main Application Scenarios

1. Development of bridging molecules for tumour immunology

One end of the bispecific aptamer recognizes tumour‑cell surface antigens. The other end binds receptors on immune‑cell surfaces. Artificial immune synapses form between tumour cells and T or NK cells to trigger tumour‑killing responses. This represents a popular direction for pre‑clinical tumour‑immunology research. Most aptamers of this type come from modular assembly of cell‑targeting aptamers obtained by SELEX [1].

2. Multi‑target biosensing and in‑vitro diagnosis

Bispecific aptamers serve as recognition elements. They capture two biomarkers within one sample simultaneously. Dual‑recognition logic greatly cuts false‑positive rates. They suit combined detection of tumour markers and inflammatory factors for building fluorescent and electrochemical biosensors.

3. Targeted drug delivery and protein degradation

One aptamer module binds disease‑related target proteins. The other engages lysosomal receptors. Target proteins get shuttled into lysosomes for degradation. Alternatively, one module homes toward lesion sites while the other anchors drug carriers. Precise drug delivery gets realised and expands application boundaries for nucleic‑acid targeting tools.

4. Molecular interaction and basic biochemical research

Bispecific aptamers work as molecular cross‑linking tools. They pull two protein molecules into close proximity. Researchers study protein‑protein contacts and dissect cellular signalling pathways. They provide new tool molecules for fundamental molecular‑biology work.

五、Bispecific Aptamer‑Related Services of KMD‑Bioscience

1. Experimental scheme design

Evaluate properties of the two targets. Decide screening pipelines, either modular assembly or Toggle‑SELEX. Design gradient groups of linkers.

2. Mono‑target aptamer screening

Select magnetic‑bead SELEX, Cell‑SELEX or CE‑SELEX according to target characteristics. Complete mono‑target aptamer selection, NGS analysis and preliminary affinity screening.

3. Bispecific aptamer construction

Synthesize multiple candidate bispecific aptamer variants with diverse linkers. Carry out sequence truncation and optimization.

4. Multi‑dimensional activity validation

Test binding performance toward each target separately. Measure protein‑level affinity with SPR. Verify cell‑surface binding via flow cytometry. Run cell‑based functional evaluation.

5. Sample delivery

Deliver complete experimental reports, raw sequencing datasets and synthesized bispecific aptamer samples. Support follow‑up chemical modification work.

Q：During bispecific aptamer validation, binding activity drops sharply for one target. What are the main causes？

1. Improper linker length or flexibility creates steric hindrance. Two aptamer modules interfere with each other and disrupt the three‑dimensional folding of one functional unit, so it loses binding capacity.

2. Original mono‑target aptamers carry redundant sequences. Intramolecular mis‑pairing occurs after assembly and prevents functional structure formation.

3. Two target molecules have large spatial dimensions. The bispecific construct cannot accommodate both macromolecular targets at the same time, giving rise to steric repulsion.

4. Buffer conditions influence nucleic‑acid folding. Assembled constructs become more sensitive toward ionic environments.

参考文献：

[1]Thomas BJ, Porciani D, Burke DH. Cancer immunomodulation using bispecific aptamers. Mol Ther Nucleic Acids. 2022;27:894-915. Published 2022 Jan 10. doi:10.1016/j.omtn.2022.01.008

[2]Kong HY, Byun J. Nucleic Acid aptamers: new methods for selection, stabilization, and application in biomedical science. Biomol Ther (Seoul). 2013;21(6):423-434. doi:10.4062/biomolther.2013.085

[3]Shraim AS, Abdel Majeed BA, Al-Binni MA, Hunaiti A. Therapeutic Potential of Aptamer-Protein Interactions. ACS Pharmacol Transl Sci. 2022;5(12):1211-1227. Published 2022 Nov 4. doi:10.1021/acsptsci.2c00156

[4]Guo R, Chen X, Ying L, et al. A novel bispecific aptamer targeting LAG3 and HER2 enhances T cell-mediated immunotherapy against HER2-positive cancer cells. Front Immunol. 2025;16:1557910. Published 2025 Jul 21. doi:10.3389/fimmu.2025.1557910