小分子靶标的非固定化核酸适配体筛选技术研究进展

doi:10.3724/SP.J.1123.2024.04012

摘要/Abstract

摘要：

核酸适配体是通过指数富集配体系统进化(SELEX)技术获得的一段核糖核酸(RNA)或单链脱氧核糖核酸(ssDNA),可用于靶标的特异性识别。核酸适配体具有易于化学合成和修饰、高热稳定性、低毒性与低免疫原性等优势。小分子靶标的核酸适配体在新药研发、肿瘤治疗、疾病诊断、超快速灵敏检测、环境污染监测和毒品检测等领域具有广泛的应用前景。然而,小分子因结构简单、分子质量小且可供核酸结合的基团有限,导致靶向小分子的适配体结合不稳定,为适配体的筛选与传感器的开发带来了巨大挑战。高效的筛选技术是获取高性能适配体的关键,目前适用于小分子靶标的核酸适配体筛选技术主要分为3类:基于靶标固定的筛选技术、基于核酸库固定的筛选技术以及靶标非固定化的筛选技术。与其他两种筛选技术相比,靶标非固定化的筛选技术所需的筛选轮数更少且获得的适配体亲和力更高(通常在nmol/L水平)。本文总结了小分子靶标的非固定化核酸适配体筛选技术(包括氧化石墨烯(GO)-SELEX、毛细管电泳(CE)-SELEX和纳米金辅助(GNP)-SELEX )的原理、优缺点及应用进展。此外,本文总结了在适配体特异性评价中对照靶标的选择策略。

关键词: 指数富集配体系统进化技术, 小分子靶标, 非固定化核酸适配体筛选技术, 综述

Abstract:

Aptamers obtained through systematic evolution of ligands by exponential enrichment (SELEX) techniques are single stranded deoxyribonucleic acid (ssDNA) or RNA molecules capable of specifically recognizing target molecules. Such aptamers are easily chemically synthesized and modified, highly thermally stable, and are low toxicity and low immunogenicity. Aptamers that target small molecules have broad applications prospects for the development of new drugs, treating tumors, diagnosing diseases, monitoring environmental pollution, detecting drugs, and in ultrafast and sensitive detection applications. However, the simple structures and low molecular masses of small molecules, along with the limited number of binding groups available for interacting with nucleic acids lead to unstable aptamer-small molecule binding, which poses significant challenges for aptamer screening and sensor development. Efficient screening techniques are crucial for identifying aptamers with excellent performance characteristics. At present, the aptamer screening techniques suitable for small-molecule targets are mainly divided into three categories: target-immobilized-based screening technique, nucleic acid library-immobilized-based screening technique, and target-non-immobilized screening technique. Among them, target-non-immobilized screening technique require fewer screening rounds and result in aptamers with superior (typically nmol/L level) affinities. This paper summarized non-immobilized aptamer screening techniques for small-molecule targets, including principle, advantages, disadvantages and application progress associated with graphene oxide (GO)-SELEX, capillary electrophoresis (CE)-SELEX, and gold nanoparticle-assisted (GNP)-SELEX techniques. In addition, strategies for selecting control targets in aptamer-specific evaluation were summarized.

Key words: systematic evolution of ligands by exponential enrichment (SELEX) techniques, small molecule targets, non-immobilized aptamer screening techniques, review

中图分类号:

O658

胡洋洋, 杨歌, 屈锋. 小分子靶标的非固定化核酸适配体筛选技术研究进展[J]. 色谱, 2025, 43(4): 297-308.

HU Yangyang, YANG Ge, QU Feng. Research advances in non-immobilized aptamer screening techniques for small-molecule targets[J]. Chinese Journal of Chromatography, 2025, 43(4): 297-308.

图/表 4

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Type	Targets	Aptamer sequences (5'-3')	Screening round	Affinity determina- tion methods	K_D/ (nmol/L)
Pesticides	tebuconazole, inaben- fide, mefenacet^[16]	CGTACGGAATTCGCTAGCAGCGTCCACGAGTGTGG- TGTGGATCCGAGCTCCACGTG, etc.	5	isothermal titration calorimetry (ITC)	10.00-100.00
	glyphosate (GlyP)^[17]	GGACAGCTGGCCGCGTAGCGAGACACGTACAAGG- TACTATACGGCTGGCATATGTATCTG	8	fluorescence assay	30.73±1.25^*
	thiamethoxam (TMX)^[18]	GTCGACGGATCCACCGACCATGCAAAGATGCACAA- AAACGACAGCAGCTGCAGGTCGAC	9	gold nanoparticle colorimetric assay	118.34±13.85^*
Toxins	T2 toxin^[19]	GTATATCAAGCATCGCGTGTTTACACATGCGAGAG- GTGAA	10	fluorescence assay	20.80±3.10^*
	gonyautoxin 1/4 (GTX1/4)^[20]	AACCTTTGGTCGGGCAAGGTAGGTT	8	biolayer interfer- ometry (BLI)	21.90
	domoic acid (DA)^[21]	AAAAATAATTTAAATTTTCTACCCAATGCTTTTCGC- ATAA	16	fluorescence assay	62.07±19.97^*
	tetrodotoxin (TTX)^[21]	TCAAATTTTCGTCTACTCAATCTTTCTGTCTTATC	16	fluorescence assay	44.12±15.38^*
	saxitoxin (STX)^[21]	CTTTTTACAAAATTCTCTTTTTACCTATATTATGAA- CAGA	16	fluorescence assay	61.44±23.18^*
	gliotoxin^[22]	CATGTGTCCACATGGAGGTGACCT	8	BLI, ITC	196.00
	okadaic acid^[23]	ATTTGACCATGTCGAGGGAGACGCGCAGTCGCTAC- CACCT	8	fluorescence assay	5.63±3.55^*
Antibiotics	penicillin G (Pen G)^[24]	CACCAGTCAGACAGCACGGTGACTGGAGTGACGTC- GGTACCTGAGATCGAGTGACGTCGGTACCTG	9	electrochemical sensing	105.15±1.94^*
	patulin^[25]	GGCCCGCCAACCCGCATCATCTACACTGATATTTTA- CCTT	15	fluorescence assay	21.83±5.02^*
	ofloxacin^[26]	TGGCGCTTAGGTGTAATAACCTGAGGACGGCTTGG, TGGTTAAACCACGGTGAACCACTGCGCAGTAGGTC	7	balanced filtration	130.10, 159.10
Drugs	ractopamine (RAC)^[27]	AGCAGCACAGAGGTCAGATGGTCTCTACTAAAAGT- TTTGATCATACCGTTCACTAATTGACCTATGCGTGC- TACCGTGAA	16	GO adsorption	54.22±8.02^*
	synthetic cannabinoids (SCs)^[28]	C6-NH2-AGGAATTCAGATCTCCCTGCAGTGGTGTTC- AATGTTTTTGTGCTGTTCTGTACTGGCGCCTCGAGG- AGCTCAGGATCCCG-SH	5	electrochemical sensing	-
	sulfamethazine (SMZ)^[29]	CGTTAGACG	7	fluorescence assay	24.60
	ephedrine^[30]	TCCGTCGGCGGCGGCCCCTTCCTACAGCTTTCCCG- GTCGC	10	ITC	2.86±0.24^*
	tramadol (TH)^[31]	CTTAAACCTGGTCGGATAGTCTTCGAGACTCGCGG- TCGCATTT	10	fluorescence assay	178.40
	L-carnitine^[32]	ACCTTGCGTGCTCACGGCAGCCTCTCGGACAGCCC- TGTGT	13	resonance rayleigh scattering (RRS)	-
Biological metabolites	β-carotene^[33]	CAGCTCAGAAGCTTGATCCTCCCACAATTATCACGT- AGTGTGCGGGTCACGCAATCTGACGACTCGAAGTC- GTGCATCTG	6	fluorescence assay	5.04±1.99^*
	25-hydroxyvitamin D3^[34]	AGCAGCACAGAGGTCATGGGGGGTGTGACTTTGGT- GTGCCTATGCGTGCTACGGAA	4	ITC	11.00
	sarcosine^[35]	TAGGGAAGAGAAGGACATATGATGTGCCGCGCTT- CCCTTGCCGCTCAAAACAGACCACCCACTTTGACT- AGTACATGACCACTTGA	8	fluorescence assay	0.33±0.05^*
Organic pollutant	nonylphenol (NP)^[36]	ATGCGGATCCCGCGCGGCCGGCCAGTGCGCGAAG- CTTGCGC	5	gold nanoparticle colorimetric assay	194.20±65.90^*

Type	Targets	Aptamer sequences (5'-3')	Screening round	Affinity determina- tion methods	K_D/ (nmol/L)
Pesticides	tebuconazole, inaben- fide, mefenacet^[16]	CGTACGGAATTCGCTAGCAGCGTCCACGAGTGTGG- TGTGGATCCGAGCTCCACGTG, etc.	5	isothermal titration calorimetry (ITC)	10.00-100.00
	glyphosate (GlyP)^[17]	GGACAGCTGGCCGCGTAGCGAGACACGTACAAGG- TACTATACGGCTGGCATATGTATCTG	8	fluorescence assay	30.73±1.25^*
	thiamethoxam (TMX)^[18]	GTCGACGGATCCACCGACCATGCAAAGATGCACAA- AAACGACAGCAGCTGCAGGTCGAC	9	gold nanoparticle colorimetric assay	118.34±13.85^*
Toxins	T2 toxin^[19]	GTATATCAAGCATCGCGTGTTTACACATGCGAGAG- GTGAA	10	fluorescence assay	20.80±3.10^*
	gonyautoxin 1/4 (GTX1/4)^[20]	AACCTTTGGTCGGGCAAGGTAGGTT	8	biolayer interfer- ometry (BLI)	21.90
	domoic acid (DA)^[21]	AAAAATAATTTAAATTTTCTACCCAATGCTTTTCGC- ATAA	16	fluorescence assay	62.07±19.97^*
	tetrodotoxin (TTX)^[21]	TCAAATTTTCGTCTACTCAATCTTTCTGTCTTATC	16	fluorescence assay	44.12±15.38^*
	saxitoxin (STX)^[21]	CTTTTTACAAAATTCTCTTTTTACCTATATTATGAA- CAGA	16	fluorescence assay	61.44±23.18^*
	gliotoxin^[22]	CATGTGTCCACATGGAGGTGACCT	8	BLI, ITC	196.00
	okadaic acid^[23]	ATTTGACCATGTCGAGGGAGACGCGCAGTCGCTAC- CACCT	8	fluorescence assay	5.63±3.55^*
Antibiotics	penicillin G (Pen G)^[24]	CACCAGTCAGACAGCACGGTGACTGGAGTGACGTC- GGTACCTGAGATCGAGTGACGTCGGTACCTG	9	electrochemical sensing	105.15±1.94^*
	patulin^[25]	GGCCCGCCAACCCGCATCATCTACACTGATATTTTA- CCTT	15	fluorescence assay	21.83±5.02^*
	ofloxacin^[26]	TGGCGCTTAGGTGTAATAACCTGAGGACGGCTTGG, TGGTTAAACCACGGTGAACCACTGCGCAGTAGGTC	7	balanced filtration	130.10, 159.10
Drugs	ractopamine (RAC)^[27]	AGCAGCACAGAGGTCAGATGGTCTCTACTAAAAGT- TTTGATCATACCGTTCACTAATTGACCTATGCGTGC- TACCGTGAA	16	GO adsorption	54.22±8.02^*
	synthetic cannabinoids (SCs)^[28]	C6-NH2-AGGAATTCAGATCTCCCTGCAGTGGTGTTC- AATGTTTTTGTGCTGTTCTGTACTGGCGCCTCGAGG- AGCTCAGGATCCCG-SH	5	electrochemical sensing	-
	sulfamethazine (SMZ)^[29]	CGTTAGACG	7	fluorescence assay	24.60
	ephedrine^[30]	TCCGTCGGCGGCGGCCCCTTCCTACAGCTTTCCCG- GTCGC	10	ITC	2.86±0.24^*
	tramadol (TH)^[31]	CTTAAACCTGGTCGGATAGTCTTCGAGACTCGCGG- TCGCATTT	10	fluorescence assay	178.40
	L-carnitine^[32]	ACCTTGCGTGCTCACGGCAGCCTCTCGGACAGCCC- TGTGT	13	resonance rayleigh scattering (RRS)	-
Biological metabolites	β-carotene^[33]	CAGCTCAGAAGCTTGATCCTCCCACAATTATCACGT- AGTGTGCGGGTCACGCAATCTGACGACTCGAAGTC- GTGCATCTG	6	fluorescence assay	5.04±1.99^*
	25-hydroxyvitamin D3^[34]	AGCAGCACAGAGGTCATGGGGGGTGTGACTTTGGT- GTGCCTATGCGTGCTACGGAA	4	ITC	11.00
	sarcosine^[35]	TAGGGAAGAGAAGGACATATGATGTGCCGCGCTT- CCCTTGCCGCTCAAAACAGACCACCCACTTTGACT- AGTACATGACCACTTGA	8	fluorescence assay	0.33±0.05^*
Organic pollutant	nonylphenol (NP)^[36]	ATGCGGATCCCGCGCGGCCGGCCAGTGCGCGAAG- CTTGCGC	5	gold nanoparticle colorimetric assay	194.20±65.90^*

Target	Screening steps	Aptamer sequence (5'-3')	Screening round	Affinity determination method	K_D/ (nmol/L)
N-Methyl mesopor- phyrin IX (NMM)^[55]	positive selection	AGCAGCACAGAGGTCAGATGTGCTCGA- CTATTGAGTCAGGGGGTTGGGGCAACA- ATAAGCCCTATGCGTGCTACCGTGAA	3	normalized fluorescence intensity determination	1200.0±100.0^*
Virginiamycin-M₁ (VGM-M₁)^[64]	positive selection	GCAGAGGGACAGGGAAGATGTAGACAT- CTGTGCTCTTCGC	4	CE-ultraviolet (UV)	49.0
Histone H4-K16Ac^[65]	counter and positive selection	AGACGTAAGTTAATTGGACTTGGTCGTG- TGCGGCACAGCGATTGAAAT	4	surface plasmon resonance (SPR)	47.0±24.0^*
Swine anaphylatoxin (C5a)^[66]	positive selection	TCGCTGTAGCTACAGGGTTTACCCGGTT- GGATGGAT	6	SPR	4000.0
Glycosylated VEGF peptide fragment^[67]	positive and counter selection	GCACCCTAATGTGCAAGCTAATGCGGAA- TGGGGTCGGTTT	8	surface plasmon reso- nance imaging (SPRI)	2500.0±400.0^*
Diethyl thiophosphate (DETP)^[68]	positive selection	GACGGGAGCTTGACACAGTCATTCCTTT- CTAGATGGTGGTT	-	gold nanoparticle colori- metric assay	103.0±14.0^*
Clenbuterol hydro- chloride (Clen)^[69]	positive selection	AGGGCCTGGGCTGTTGAGGCAACGTCG- GTTTGTTTATTAA	3	capillary electrophoresis- laser induced fluorescence (LIF)	931.5
Neuropeptide Y (NPY)^[70]	positive selection	AGCAGCACAGAGGTCAGATGCAAACCA- CAGCCTGATGGTTAGCGTATGTCATTTA- CGGACCTATGCGTGCTACCGTGAA	4	affinity capillary electro- phoresis (ACE)	800.0

Target	Screening steps	Aptamer sequence (5'-3')	Screening round	Affinity determination method	K_D/ (nmol/L)
N-Methyl mesopor- phyrin IX (NMM)^[55]	positive selection	AGCAGCACAGAGGTCAGATGTGCTCGA- CTATTGAGTCAGGGGGTTGGGGCAACA- ATAAGCCCTATGCGTGCTACCGTGAA	3	normalized fluorescence intensity determination	1200.0±100.0^*
Virginiamycin-M₁ (VGM-M₁)^[64]	positive selection	GCAGAGGGACAGGGAAGATGTAGACAT- CTGTGCTCTTCGC	4	CE-ultraviolet (UV)	49.0
Histone H4-K16Ac^[65]	counter and positive selection	AGACGTAAGTTAATTGGACTTGGTCGTG- TGCGGCACAGCGATTGAAAT	4	surface plasmon resonance (SPR)	47.0±24.0^*
Swine anaphylatoxin (C5a)^[66]	positive selection	TCGCTGTAGCTACAGGGTTTACCCGGTT- GGATGGAT	6	SPR	4000.0
Glycosylated VEGF peptide fragment^[67]	positive and counter selection	GCACCCTAATGTGCAAGCTAATGCGGAA- TGGGGTCGGTTT	8	surface plasmon reso- nance imaging (SPRI)	2500.0±400.0^*
Diethyl thiophosphate (DETP)^[68]	positive selection	GACGGGAGCTTGACACAGTCATTCCTTT- CTAGATGGTGGTT	-	gold nanoparticle colori- metric assay	103.0±14.0^*
Clenbuterol hydro- chloride (Clen)^[69]	positive selection	AGGGCCTGGGCTGTTGAGGCAACGTCG- GTTTGTTTATTAA	3	capillary electrophoresis- laser induced fluorescence (LIF)	931.5
Neuropeptide Y (NPY)^[70]	positive selection	AGCAGCACAGAGGTCAGATGCAAACCA- CAGCCTGATGGTTAGCGTATGTCATTTA- CGGACCTATGCGTGCTACCGTGAA	4	affinity capillary electro- phoresis (ACE)	800.0

Target	Nucleic acid library type	Aptamer sequences (5'-3')	Screening round	Affinity determination method	K_D/ (μmol/L)
Dichlorvos (DV)^[78]	DNA	GGAAGAACATGTGAGCAAAAGGCCAGCAAAAGG- CCAGGAACCGTAAAAAGGCCGCGTTGCTGGC	8	ITC	0.850
Amyloid β-peptide (Aβ)^[79]	RNA	UAGCGUAUGCCACUCUCCUGGGACCCCCCGCCG- GAUGGCCA, UUUGGGGUGUCGGGCGAUUUUUA- GGGUUGGGCCAGGCCGU	9	Fluorescence anisotropy measurements	21.6, 10.9
Zinc(II)-protoporphy- rin IX (ZnPPIX)^[80]	DNA	GGCGGGGGGTTGCTCTACTTGATGATCTGCGCTA- TCGCCGT	11	fluorescence assay	9.53±1.86^*
Brassinolide (BL)^[81]	DNA	GCGGATCCCGCGCCGTGCAGAGGGAGACCGC	9	gold nanoparticle colorimetric assay	0.0173
Bisphenol A (BPA)^[81]	DNA	GGACGCGCGAAGAATATAAGGTGGCCTGGCCGC- GCG	11	gold nanoparticle colorimetric assay	0.0379