Boron-containing aptamers to ATP

Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5′-(α-P-borano) triphosphate (bG) for GTP or uridine 5′-(α-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the ζ-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some ζ-fold aptamers, while the bG selection yielded none of this type. Non-ζ aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.     

综述与专论: 基于核酸适配体传感器的即时检测（POCT）技术

即时检测（point-of-care testing，POCT）是一种检测成本低、检测速度快、准确度高、能自我采样获得临床诊断结果的新型诊断技术。该技术在临床诊断、病情监控与疫情防控等领域发挥了重要作用。核酸适配体是一种能够特异性识别多种靶标的分子探针，具有易合成、批间差异小、易实现信号放大等突出优势，是生物医学传感器中重要的分子识别元件。本文概述了核酸适配体探针的现有筛选方法和进展，总结了核酸适配体POCT传感器信号放大策略，着重介绍了各类核酸适配体传感器在POCT领域的应用现状，并对核酸适配体POCT传感器的发展前景进行了展望。     

Selection and evolution of NTP-specific aptamers

ATP occupies a central position in biology, for it is both an elementary building block of RNA and the most widely used cofactor in all living organisms. For this reason, it has been a recurrent target for in vitro molecular evolution techniques. The exploration of ATP-binding motifs constitutes both an important step in investigating the plausibility of the ‘RNA world’ hypothesis and a central starting point for the development of new enzymes. To date, only two RNA motifs that bind ATP have been characterized. The first one is targeted to the adenosine moiety, while the second one recognizes the ‘Hoogsteen’ face of the base. To isolate aptamers that bind ATP in different orientations, we selected RNAs on an affinity resin that presents ATP in three different orientations. We obtained five new motifs that were characterized and subsequently submitted to a secondary selection protocol designed to isolate aptamers specific for cordycepin. Interestingly, all the ATP-binding motifs selected specifically recognize the sugar-phosphate backbone region of the nucleotides. Three of the aptamers show some selectivity for adenine derivatives, while the remainder recognize any of the four nucleotides with similar efficiency. The characteristics of these aptamers are discussed along with implications for in vitro molecular evolution.     

信号适体的设计策略及应用

信号适体兼具有分子识别和信号转导的功能.从随机寡核苷酸库中筛选出的适体,要经过合理设计和筛选后修饰,才具备信号转导功能.信号适体可分为标记和非标记两大类.本文着重介绍荧光标记信号适体的设计策略,包括基于荧光偏振分析标记一个荧光基团,及基于荧光共振能量转移同时标记荧光基团、淬灭基团,或两个荧光基团的信号适体(包括分子信标适体、结构转换和原位标记信号适体).非标记信号适体的设计,有嵌合法、置换法、光转换复合物法,及适体－多聚物偶联法.此外,亦可直接从体外筛选出信号适体.信号适体的诸多优点利于其用于生物传感器及均相液相中实时蛋白识别与定量分析.     

Analytical potential of gold nanoparticles in functional aptamer-based biosensors

Gold nanoparticles (AuNPs) exhibit many predominant capabilities such as high biocompatibility, chemical stability, strong localized surface plasmon resonance absorption, and high extinction coefficient in the visible region. These properties have enabled the extensive use of AuNPs in optical and electrochemical biosensors. As a kind of functional nucleic acids, aptamers can be considered as a valid alternative to antibodies or other bio-receptors and have been widely employed to develop novel biosensors. We are summarizing here the state of the art of AuNP-based biosensors that use functional aptamers as molecular recognition elements. In many cases, AuNPs themselves can be used as a probe for detection, such as various colorimetric aptasensors and fluorescent aptasensors. They also can be used as probe vectors to enlarge detection signals and to increase the number of conceivable substrates in electrochemical aptasensors.     

综述与专论: 基于核酸适配体的细胞外囊泡分离分析方法

细胞外囊泡通过参与细胞间通讯，在诸多生理病理过程中发挥着重要作用。因此，细胞外囊泡的分离分析对理解其生物学功能以及发展基于囊泡的疾病诊疗方法具有重要价值。细胞外囊泡的高效分离以及高灵敏可靠检测很大程度上取决于识别配体。核酸适配体是一类高效、特异结合其靶标分子的单链寡核苷酸。核酸适配体的易修饰和可程序化设计等特征，使其成为细胞外囊泡分离和分析的理想识别配体。为提高细胞外囊泡的分离效率，研究者们提出多种策略用于提升核酸适配体的亲和力，以及界面与细胞外囊泡的接触几率。此外，分离不同亚型的细胞外囊泡有助于理解细胞外囊泡的生物学意义。在细胞外囊泡分析方面，根据核酸适配体与细胞外囊泡识别信号的转导方式不同，分为电化学、可视化、表面增强拉曼光谱、荧光法等方法。本文综述了核酸适配体的筛选以及其在细胞外囊泡分离和分析中的最新进展、挑战及未来方向。     

CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface -Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.     

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.     

Regulation of uridine kinase quaternary structure. Dissociation by the inhibitor CTP

Uridine kinase from mouse Ehrlich ascites cells can exist in a variety of different aggregation states, from monomer up to aggregates that may contain 32 or more subunits. With very crude enzyme preparations, uridine kinase activity is always associated with several different coexisting molecular weight species. Changes in the aggregation state are produced in the presence of normal effectors (orthophosphate, ATP and CTP) at physiological concentrations. With uridine kinase that has been purified 9,000-fold, enzyme activity is associated with only a single molecular weight species, but is still responsive to the same physiological effectors. In the presence of orthophosphate, uridine kinase has a molecular weight of 380,000 (appropriate for a dodecamer). In the presence of CTP, the enzyme dissociates with concomitant loss of activity. The dissociated enzyme can be reassociated to the native size. These results imply that alteration of the enzyme's quaternary structure by normal effectors constitutes a mechanism for regulating uridine kinase activity in vivo.     

DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.     

Molecular signaling aptamers for real-time fluorescence analysis of protein

Aptamers are a new class of nucleic acids that are selected in vitro for binding target molecules with high affinity and selectivity. They are promising protein-binding molecular probes that rival conventional antibodies for protein analysis. There have been recent advances in the development of molecular signaling aptamers that can transduce target protein binding to sensitive fluorescence signal changes. This facilitates the real time protein monitoring in homogenous solution as well as potentially in vivo. Different signaling strategies of using dual labeled aptamers based on fluorescence resonance energy transfer (FRET), one fluorophore labeled aptamers based on fluorescence anisotropy assay, or other label-free aptamers are reviewed.     

A fluorescent aptasensor for analysis of adenosine triphosphate based on aptamer–magnetic nanoparticles and its single‐stranded complementary DNA labeled carbon dots

A new fluorimetric aptasensor was designed for the determination of adenosine triphosphate (ATP) based on magnetic nanoparticles (MNPs) and carbon dots (CDs). In this analytical strategy, an ATP aptamer was conjugated on MNPs and a complementary strand of the aptamer (CS) was labeled with CDs. The aptamer and its CS were hybridized to form a double helical structure. The hybridized aptamers could be used for the specific recognition of ATP in a biological complex matrix using a strong magnetic field to remove the interfering effect. In the absence of ATP, no CDs–CS could be released into the solution and this resulted in a weak fluorescence signal. In the presence of ATP, the target binds to its aptamer and causes the dissociation of the double helical structure and liberation of the CS, such that a strong fluorescence signal was generated. The increased fluorescence signal was proportional to ATP concentration. The limit of detection was estimated to be 1.0 pmol L^–1 with a dynamic range of 3.0 pmol L^–1 to 5.0 nmol L^–1. The specific aptasensor was applied to detect ATP in human serum samples with satisfactory results. Moreover, molecular dynamic simulation (MDS) studies were used to analyze interactions of the ATP molecule with the aptamer.     

Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

Background

Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor.

Methodology/Principal Findings

We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment.

Conclusion/Significance

The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma.     

细胞SELEX技术用于胰腺癌细胞特异性适配体的筛选及鉴定

细胞SELEX是目前常用的筛选细胞特异性适配体的技术。胰腺癌细胞特异性适配体在胰腺癌的诊断和治疗中有巨大的应用潜力。本研究拟通过该技术获得特异性识别胰腺癌PANC-1细胞的适配体,并对所筛选的适配体进行功能鉴定。本研究以胰腺癌PANC-1细胞为正筛细胞,正常胰腺导管上皮细胞HPDE6-C7为负筛细胞,通过磁珠法细胞SELEX技术进行筛选。经过12轮筛选,对筛选文库进行PCR扩增、质粒转染、单克隆挑选及测序,获得2条适配体Apt-5和Apt-12。流式细胞术检测发现,适配体Apt-5和Apt-12可特异性识别PANC-1细胞,其Kd值分别为8.27±2.10 nmol/L和8.88±2.51 nmol/L,Kd值均处于纳摩尔级别。通过RNA结构预测,2条适配体的二级结构均为茎-环结构。细胞免疫荧光验证了适配体的结合部位为细胞膜表面。本研究表明,通过磁珠法细胞SELEX技术,成功获得可特异性识别胰腺癌PANC-1细胞的适配体。该适配体有望成为胰腺癌诊断和治疗中的特异性分子靶向剂。     

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.     

In vitro selection of DNA aptamers binding pesticide fluoroacetamide

Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated K_D values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.     

DNA aptamers as molecular probes for colorectal cancer study

Background

Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s) underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development.

Methodology and Results

Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX) and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh), nor do they recognize most other cancer cell lines tested.

Conclusion/Significance

The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers.     

Selection and Identification of Aptamers Against B Cell Activating Factor

Systemic lupus erythematosus (SLE) is the most common autoimmune disease in China. B cell activating factor (BAFF) is an important target for the treatment and detection of SLE. It is of great significance to develop novel molecular recognition elements with high affinity for BAFF. In this study, artificial nucleic acid aptamers against BAFF were screened from a 78 nt single-stranded DNA random library by systematic evolution of ligands exponential enrichment (SELEX) in vitro based on several selection and amplification steps. Through ten rounds of selection, the aptamers with high specificity and affinity for BAFF were identified. After high-throughput sequencing, several aptamers were selected and further examined for binding affinity and specificity. The investigation by dot blotting, Eastern blotting analyses and enzyme-linked oligonucleotide assay (ELONA) showed that the aptamers Apt 7 and Apt 12 with dissociation constants of 241.00±19.75 nmol/L and 413.51±46.94 nmol/L were able to recognize BAFF specifically. After molecular docking analysis, Apt 7 was truncated to Apt 7～1, and the dissociation constant was 192.10±28.61 nmol/L. A sandwich ELONA using Apt 7～1 and BAFF antibodies was established to detect BAFF. The detection limit was estimated to be 0.227 nmol/L. This study provides new molecular recognition elements for the detection of BAFF and the study of antagonists.     

Selection of Aptamers for Mature White Adipocytes by Cell SELEX Using Flow Cytometry

Background

Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited.

Methods and Results

We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes.

Conclusions

These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity.