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Fulltext Analytical and Clinical Evaluation of a TaqMan Real-Time PCR Assay for the Detection of Chikungunya Virus

Andrew A, Citartan M, Wong KA, Tang TH, Magdline Sia Henry S, Ch'ng ES

Microbiol Spectr, 2023 Aug 17;11(4):e0008823.
PMID: 37272795 DOI: 10.1128/spectrum.00088-23

Due to the general symptoms presented by the Chikungunya virus (CHIKV)-infected patients, a laboratory test is needed to differentiate CHIKV from other viral infections. The reverse transcription-quantitative real-time PCR (RT-qPCR) is a rapid and sensitive diagnostic tool, and several assays have been developed for detecting and quantifying CHIKV. Since real-time amplification efficiency varies within and between laboratories, an assay must be validated before being used on patient samples. In this study, the diagnostic performance of a TaqMan RT-qPCR assay was evaluated using synthetic RNA and archived patient samples. The cutoff quantification cycle (Cq) value for the assay was determined by experimental evidence. We found the in-house assay was highly sensitive, with a detection limit of 3.95 RNA copies/reaction. The analytical specificity of the assay was 100%. The analytical cutoff Cq value was 37, corresponding to the mean Cq value of the detection limit. Using archived samples characterized previously, the sensitivity and specificity of the assay were 76% and 100%, respectively. The in-house assay was also compared with a commercial assay, and we found that the in-house assay had higher sensitivity. Although further evaluation with prospective patient samples is needed in the future, this validated RT-qPCR was sensitive and specific, which shows its potential to detect CHIKV in clinical samples. IMPORTANCE Chikungunya virus causes chikungunya fever, a disease characterized by fever, rash, and joint pain. In the early phase of infection, chikungunya fever is always misdiagnosed as other arbovirus infections, such as dengue. Laboratory tests such as RT-qPCR are therefore necessary to confirm CHIKV infection. We evaluated the performance of an in-house RT-qPCR assay, and our study shows that the assay could detect CHIKV in clinical samples. We also show the cutoff determination of the assay, which provides important guidance to scientists or researchers when implementing a new RT-qPCR assay in a laboratory.
In-silico selection employing rigid docking and molecular dynamic simulation in selecting DNA aptamers against androgen receptor

Thevendran R, Tang TH, Citartan M

Biotechnol J, 2023 Apr;18(4):e2200092.
PMID: 36735817 DOI: 10.1002/biot.202200092

Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformations and high GC content. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA), which includes the measurement of their binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer, termed ARapt1 with the estimated Kd value of 5.5 ± 3 nm, was chosen as the most prominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.
Fulltext mRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles

Azhar NA, Abu Bakar SA, Citartan M, Ahmad NH

World J Hepatol, 2023 Mar 27;15(3):393-409.
PMID: 37034237 DOI: 10.4254/wjh.v15.i3.393

BACKGROUND: The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy. However, the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles (AgNPs) makes its precise mechanisms of action unclear.
AIM: To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G. Don (C. roseus)-AgNPs.
METHODS: The proliferative activity of hepatocellular carcinoma (HepG2) and normal human liver (THLE3) cells treated with C. roseusAgNPs were measured using MTT assay. The RNA samples were extracted and sequenced using BGIseq500 platform. This is followed by data filtering, mapping, gene expression analysis, differentially expression genes analysis, Gene Ontology analysis, and pathway analysis.
RESULTS: The mean IC50 values of C. roseusAgNPs on HepG2 was 4.38 ± 1.59 μg/mL while on THLE3 cells was 800 ± 1.55 μg/mL. Transcriptome profiling revealed an alteration of 296 genes. C. roseusAgNPs induced the expression of stress-associated genes such as MT, HSP and HMOX-1. Cellular signalling pathways were potentially activated through MAPK, TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest. The alteration of ARF6, EHD2, FGFR3, RhoA, EEA1, VPS28, VPS25, and TSG101 indicated the uptake of C. roseus-AgNPs via both clathrin-dependent and clathrin-independent endocytosis.
CONCLUSION: This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells. The cytotoxicity effect is mediated by the aberrant gene alteration, and more interestingly the unique selective antiproliferative properties indicate the C. roseusAgNPs as an ideal anticancer candidate.
Generation of an RNA aptamer against LipL32 of Leptospira isolated by Tripartite-hybrid SELEX coupled with in-house Python-aided unbiased data sorting

Shien Yeoh T, Yusof Hazrina H, Bukari BA, Tang TH, Citartan M

Bioorg Med Chem, 2023 Mar 01;81:117186.
PMID: 36812779 DOI: 10.1016/j.bmc.2023.117186

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira. The major hurdle of the diagnosis of Leptospirosis lies in the issues associated with current methods of detection, which are time-consuming, tedious and the need for sophisticated, special equipments. Restrategizing the diagnostics of Leptospirosis may involve considerations of the direct detection of the outer membrane protein, which can be faster, cost-saving and require fewer equipments. One such promising marker is LipL32, which is an antigen with high amino acid sequence conservation among all the pathogenic strains. In this study, we endeavored to isolate an aptamer against LipL32 protein via a modified SELEX strategy known as tripartite-hybrid SELEX, based on 3 different partitioning strategies. In this study, we also demonstrated the deconvolution of the candidate aptamers by using in-house Python-aided unbiased data sorting in examining multiple parameters to isolate potent aptamers. We have successfully generated an RNA aptamer against LipL32 of Leptospira, LepRapt-11, which is applicable in a simple direct ELASA for the detection of LipL32. LepRapt-11 can be a promising molecular recognition element for the diagnosis of leptospirosis by targeting LipL32.
Isolation of a novel DNA aptamer against LipL32 as a potential diagnostic agent for the detection of pathogenic Leptospira

Yeoh TS, Tang TH, Citartan M

Biotechnol J, 2023 Mar;18(3):e2200418.
PMID: 36426669 DOI: 10.1002/biot.202200418

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira and for rapid diagnostics, direct detection is desirable. LipL32 protein is the most suitable biomarker for direct detection. DNA aptamers are sought to be generated against LipL32 by Systemic Evolution of Ligands via Exponential Enrichment (SELEX). LepDapt-5a is the most potent aptamer candidate among all the candidates, as determined by direct Enzyme-linked Aptasorbent Assay (ELASA). LepDapt-5a was predicted to form a G-quadruplex structure as predicted by QGRS Mapper and validated experimentally by direct ELASA. The diagnostic potential of the aptamer was further tested on a direct and sandwich ELASA platform. A LOD of 106 mL-1 and 105 mL-1 were estimated by direct and sandwich ELASA platforms, respectively, which are within the range associated with leptospiremia levels. The dot blot assay developed was able to attain a LOD of 104 CFU mL-1 against pathogenic Leptospira, which is also within the leptospiremia level. This is the first-ever DNA aptamer and hybrid-heterodimeric aptamer constructed against LipL32. The diagnostic potentiality of the LepDapt-5a DNA aptamer was proven on three major diagnostic platforms, which are direct ELASA, sandwich ELASA, and aptamer-based dot assay.
Fulltext Biocomputational Identification of sRNAs in Leptospira interrogans Serovar Lai

Tan XY, Citartan M, Chinni SV, Ahmed SA, Tang TH

Indian J Microbiol, 2023 Mar;63(1):33-41.
PMID: 37188232 DOI: 10.1007/s12088-022-01050-9

Regulatory small RNAs (sRNA) are RNA transcripts that are not translated into proteins but act as functional RNAs. Pathogenic Leptospira cause an epidemic spirochaetal zoonosis, Leptospirosis. It is speculated that Leptospiral sRNAs are involved in orchestrating their pathogenicity. In this study, biocomputational approach was adopted to identify Leptospiral sRNAs. In this study, two sRNA prediction programs, i.e., RNAz and nocoRNAc, were employed to screen the reference genome of Leptospira interrogans serovar Lai. Out of 126 predicted sRNAs, there are 96 cis-antisense sRNAs, 28 trans-encoded sRNAs and 2 sRNAs that partially overlap with protein-coding genes in a sense orientation. To determine whether these candidates are expressed in the pathogen, they were compared with the coverage files generated from our RNA-seq datasets. It was found out that 7 predicted sRNAs are expressed in mid-log phase, stationary phase, serum stress, temperature stress and iron stress while 2 sRNAs are expressed in mid-log phase, stationary phase, serum stress, and temperature stress. Besides, their expressions were also confirmed experimentally via RT-PCR. These experimentally validated candidates were also subjected to mRNA target prediction using TargetRNA2. Taken together, our study demonstrated that biocomputational strategy can serve as an alternative or as a complementary strategy to the laborious and expensive deep sequencing methods not only to uncover putative sRNAs but also to predict their targets in bacteria. In fact, this is the first study that integrates computational approach to predict putative sRNAs in L. interrogans serovar Lai.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-022-01050-9.
A comparative study of aptamer isolation by conventional and microfluidic strategies

Meng X, Wen K, Citartan M, Lin Q

Analyst, 2023 Feb 13;148(4):787-798.
PMID: 36688616 DOI: 10.1039/d2an01767a

Aptamers are single-stranded oligonucleotide molecules that bind with high affinity and specificity to a wide range of target molecules. The method of systematic evolution of ligands by exponential enrichment (SELEX) plays an essential role in the isolation of aptamers from a randomized oligonucleotide library. To date, significant modifications and improvements of the SELEX process have been achieved, engendering various forms of SELEX from conventional SELEX to microfluidics-based full-chip SELEX. While full-chip SELEX is generally considered advantageous over conventional SELEX, there has not yet been a conclusive comparison between the methods. Herein, we present a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection, and fully integrated microfluidic affinity selection and PCR amplification. Using immunoglobulin E (IgE) as a model target molecule, we compare these strategies in terms of the time and cost for each step of the SELEX process including affinity selection, amplification, and oligonucleotide conditioning. Target-binding oligonucleotides in the enriched pools are sequenced and compared to assess the relative efficacy of the SELEX strategies. We show that the microfluidic strategies are more time- and cost-efficient than conventional SELEX.
Fulltext Diagnostic accuracy of serological tests for the diagnosis of Chikungunya virus infection: A systematic review and meta-analysis

Andrew A, Navien TN, Yeoh TS, Citartan M, Mangantig E, Sum MSH, et al.

PLoS Negl Trop Dis, 2022 Feb;16(2):e0010152.
PMID: 35120141 DOI: 10.1371/journal.pntd.0010152

BACKGROUND: Chikungunya virus (CHIKV) causes febrile illnesses and has always been misdiagnosed as other viral infections, such as dengue and Zika; thus, a laboratory test is needed. Serological tests are commonly used to diagnose CHIKV infection, but their accuracy is questionable due to varying degrees of reported sensitivities and specificities. Herein, we conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of serological tests currently available for CHIKV.
METHODOLOGY AND PRINCIPAL FINDINGS: A literature search was performed in PubMed, CINAHL Complete, and Scopus databases from the 1st December 2020 until 22nd April 2021. Studies reporting sensitivity and specificity of serological tests against CHIKV that used whole blood, serum, or plasma were included. QUADAS-2 tool was used to assess the risk of bias and applicability, while R software was used for statistical analyses. Thirty-five studies were included in this meta-analysis; 72 index test data were extracted and analysed. Rapid and ELISA-based antigen tests had a pooled sensitivity of 85.8% and 82.2%, respectively, and a pooled specificity of 96.1% and 96.0%, respectively. According to our meta-analysis, antigen detection tests serve as a good diagnostic test for acute-phase samples. The IgM detection tests had more than 90% diagnostic accuracy for ELISA-based tests, immunofluorescence assays, in-house developed tests, and samples collected after seven days of symptom onset. Conversely, low sensitivity was found for the IgM rapid test (42.3%), commercial test (78.6%), and for samples collected less than seven of symptom onset (26.2%). Although IgM antibodies start to develop on day 2 of CHIKV infection, our meta-analysis revealed that the IgM detection test is not recommended for acute-phase samples. The diagnostic performance of the IgG detection tests was more than 93% regardless of the test formats and whether the test was commercially available or developed in-house. The use of samples collected after seven days of symptom onset for the IgG detection test suggests that IgG antibodies can be detected in the convalescent-phase samples. Additionally, we evaluated commercial IgM and IgG tests for CHIKV and found that ELISA-based and IFA commercial tests manufactured by Euroimmun (Lübeck, Germany), Abcam (Cambridge, UK), and Inbios (Seattle, WA) had diagnostic accuracy of above 90%, which was similar to the manufacturers' claim.
CONCLUSION: Based on our meta-analysis, antigen or antibody-based serological tests can be used to diagnose CHIKV reliably, depending on the time of sample collection. The antigen detection tests serve as a good diagnostic test for samples collected during the acute phase (≤7 days post symptom onset) of CHIKV infection. Likewise, IgM and IgG detection tests can be used for samples collected in the convalescent phase (>7 days post symptom onset). In correlation to the clinical presentation of the patients, the combination of the IgM and IgG tests can differentiate recent and past infections.
Fulltext Aptamers functionalized hybrid nanomaterials for algal toxins detection and decontamination in aquatic system: Current progress, opportunities, and challenges

Bilibana MP, Citartan M, Fuku X, Jijana AN, Mathumba P, Iwuoha E

Ecotoxicol Environ Saf, 2022 Jan 29;232:113249.
PMID: 35104779 DOI: 10.1016/j.ecoenv.2022.113249

Purification and detection of algal toxins is the most effective technique to ensure that people have clean and safe drinking water. To achieve these objectives, various state-of-the-art technologies were designed and fabricated to decontaminate and detect algal toxins in aquatic environments. Amongst these technologies, aptamer-functionalized hybrid nanomaterials conjugates have received significant consideration as a result of their several benefits over other methods, such as good controllable selectivity, low immunogenicity, and biocompatibility. Because of their excellent properties, aptamer-functionalized hybrid nanomaterials conjugates are one of several remarkable agents. Several isolated aptamer sequences for algal toxins are addressed in this review, as well as aptasensor and decontamination aptamer functionalized metal nanoparticle-derived hybrid nanocomposites applications. In addition, we present diverse aptamer-functionalized hybrid nanomaterial conjugates designs and their applications for sensing and decontamination.
Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners

Yeoh TS, Anna A, Tang TH, Citartan M

World J Microbiol Biotechnol, 2022 Jan 06;38(2):31.
PMID: 34989899 DOI: 10.1007/s11274-021-03209-w

Asymmetric PCR is one of the most utilized strategies in ssDNA generation towards DNA aptamer generation due to its low cost, robustness and the low amount of starting template. Despite its advantages, careful optimization of the asymmetric PCR is still warranted to optimize the yield of ssDNA. In this present study, we have developed an extensive optimization pipeline that involves the optimization of symmetric PCR initially followed by the optimization of asymmetric PCR. In the asymmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg2+/dNTP concentrations and the amounts of Taq Polymerase was carried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2 nM for A-PCR-lambda exonuclease and A-PCR-biotin-streptavidin-based separation, respectively. We urge to develop a separate optimization pipeline of asymmetric PCR for each different randomized ssDNA library before embarking on any SELEX studies.
The dynamicity of light-up aptamers in one-pot in vitro diagnostic assays

Citartan M

Analyst, 2021 Dec 20;147(1):10-21.
PMID: 34860215 DOI: 10.1039/d1an01690c

Light-up aptamers are aptamers that ignite the fluorescence emission of certain dyes upon binding. Widely harnessed in in vivo imaging, the binding capacity of the light-up aptamers can also be deployed in in vitro diagnostic assays, engendering a mix-and-read format. Intrigued by this, I intend to provide an overview of the various formats of diagnostic assays developed using light-up aptamers from the direct modulation of the light-up aptamers, split aptamer-based configuration, strand displacement, in vitro transcription-based one-pot diagnostic assay, CRISPR-Cas system to the measurement of the ion reliance. The incorporation of the light-up aptamers into each configuration is expounded and further supported by describing the exemplary assays developed thus far. It is anticipated that the present study can be enlightening to any researchers who aspire to embark on the development of one-pot in vitro diagnostic assays based on light-up aptamers.
Aptamers as the powerhouse of dot blot assays

Citartan M

Talanta, 2021 Sep 01;232:122436.
PMID: 34074421 DOI: 10.1016/j.talanta.2021.122436

Dot blot assays have always been associated with antibodies as the main molecular recognition element, which are widely employed in a myriad of diagnostic applications. With the rising of aptamers as the equivalent molecular recognition elements of antibodies, dot blot assays are also one of the diagnostic avenues that should be scrutinized for their amenability with aptamers as the potential surrogates of antibodies. In this review, the stepwise procedures of an aptamer-based dot blot assays are underscored before reviewing the existing aptamer-based dot blot assays developed so far. Most of the applications center on monitoring the progress of SELEX and as the validatory assays to assess the potency of aptamer candidates. For the purpose of diagnostics, the current effort is still languid and as such possible suggestions to galvanize the move to spur the aptamer-based dot blot assays to a point-of-care arena are discussed.
Aptamers isolated against mosquito-borne pathogens

Navien TN, Yeoh TS, Anna A, Tang TH, Citartan M

World J Microbiol Biotechnol, 2021 Jul 09;37(8):131.
PMID: 34240263 DOI: 10.1007/s11274-021-03097-0

Mosquito-borne diseases are a major threat to public health. The shortcomings of diagnostic tools, especially those that are antibody-based, have been blamed in part for the rising annual morbidity and mortality caused by these diseases. Antibodies harbor a number of disadvantages that can be clearly addressed by aptamers as the more promising molecular recognition elements. Aptamers are defined as single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit high binding affinity and specificity against a wide variety of target molecules based on their unique structural conformations. A number of aptamers were developed against mosquito-borne pathogens such as Dengue virus, Zika virus, Chikungunya virus, Plasmodium parasite, Francisella tularensis, Japanese encephalitis virus, Venezuelan equine encephalitis virus, Rift Valley fever virus and Yellow fever virus. Intrigued by these achievements, we carry out a comprehensive overview of the aptamers developed against these mosquito-borne infectious agents. Characteristics of the aptamers and their roles in diagnostic, therapeutic as well as other applications are emphasized.
Comparative genomic identification and characterization of npcRNA homologs in Proteus vulgaris

Kishanraj S, Sumitha S, Tang TH, Citartan M, Chinni SV

J Biosci, 2021;46.
PMID: 34845992

Proteus vulgaris is a rod-shaped Gram-negative bacterium known to be the member of Enterobacteriaceae that is able to cause disease in human being. Generally, non-protein-coding RNAs (npcRNAs) do not code for proteins, but they play a vital role in gene regulation at the RNA level including pathogenicity. The present study aims at elucidating homologous npcRNAs from other bacteria in Proteus vulgaris. A comparative genomic analysis was carried out to identify npcRNA homolog of other Enterobacteriaceae pathogens in Proteus vulgaris. A total of 231 npcRNAs previously reported in Salmonella typhi, Salmonella typhimurium and Escherichia coli were screened using BLASTn tool against Proteus vulgaris genome. Interestingly, 33 npcRNAs are homologs to Proteus vulgaris. Northern blot analysis of 6 out of 33 npcRNA candidates confirmed their expression and showed that most of them are differentially expressed during lag, exponential and stationary growth phases. This study is the first approach of identification and characterization of npcRNAs in P. vulgaris. Hence, this could be a pioneer study to further validate the regulatory functions of these npcRNAs to fill the gaps in understanding of the pathogenicity of P. vulgaris.
Unravelling the diagnostic and therapeutic potentialities of a novel RNA aptamer isolated against human pituitary tumour transforming gene 1 (PTTG1) protein

Prabu SS, Ch'ng ES, Woon PY, Chen JH, Tang TH, Citartan M

Anal Chim Acta, 2020 Nov 22;1138:181-190.
PMID: 33161980 DOI: 10.1016/j.aca.2020.09.038

Human Pituitary Tumour Transforming Gene 1 (PTTG1) is an oncoprotein involved in maintaining chromosome stability and acts as a biomarker for a panel of cancers. In this study, we endeavoured to generate an RNA aptamer against PTTG1. The RNA aptamer, SECURA-3 has an estimated equilibrium dissociation constant of 16.41 ± 6.4 nM. The aptamer was successfully harnessed in several diagnostic platforms including ELASA, aptamer-based dot blot and aptamer-based western blot. SECURA-3 was also unveiled as a potential probe that could replace its counterpart antibody in the histostaining-based detection of PTTG1 in HeLa and MCF-7 formalin-fixed paraffin-embedded cell blocks. In the aspect of therapeutics, SECURA-3 RNA aptamer demonstrates an antagonistic effect by antagonizing the interaction between PTTG1 and CXCR2, as revealed in the in vitro competitive nitrocellulose filter binding assay and dual-luciferase reporter assay in HeLa cells. As the first anti-PTTG1 aptamer, SECURA-3 RNA aptamer has immense diagnostic and therapeutic properties.
In silico molecular docking in DNA aptamer development

Navien TN, Thevendran R, Hamdani HY, Tang TH, Citartan M

Biochimie, 2020 Oct 18;180:54-67.
PMID: 33086095 DOI: 10.1016/j.biochi.2020.10.005

Aptamers are single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit binding affinity and specificity against a wide variety of target molecules. Compared to RNA aptamers, DNA aptamers are much more stable and therefore are widely adopted in a number of applications especially in diagnostics. The tediousness and rigor associated with certain steps of the SELEX intensify the efforts to adopt in silico molecular docking approaches together with in vitro SELEX procedures in developing DNA aptamers. Inspired by these endeavors, we carry out an overview of the in silico molecular docking approaches in DNA aptamer generation, by detailing the stepwise procedures as well as shedding some light on the various softwares used. The in silico maturation strategy and the limitations of the in silico approaches are also underscored.
Strategies to bioengineer aptamer-driven nanovehicles as exceptional molecular tools for targeted therapeutics: A review

Thevendran R, Sarah S, Tang TH, Citartan M

J Control Release, 2020 07 10;323:530-548.
PMID: 32380206 DOI: 10.1016/j.jconrel.2020.04.051

Aptamers are a class of folded nucleic acid strands capable of binding to different target molecules with high affinity and selectivity. Over the years, they have gained a substantial amount of interest as promising molecular tools for numerous medical applications, particularly in targeted therapeutics. However, only the different treatment approaches and current developments of aptamer-drug therapies have been discussed so far, ignoring the crucial technical and functional aspects of constructing a therapeutically effective aptamer-driven drug delivery system that translates to improved in-vivo performance. Hence, this paper provides a comprehensive review of the strategies used to improve the therapeutic performance of aptamer-guided delivery systems. We focus on the different functional features such as drug deployment, payload capacity, in-vivo stability and targeting efficiency to further our knowledge in enhancing the cell-specific delivery of aptamer-drug conjugates. Each reported strategy is critically discussed to emphasize both the benefits provided in comparison with other similar techniques and to outline their potential drawbacks with respect to the molecular properties of the aptamers, the drug and the system to be designed. The molecular architecture and design considerations for an efficient aptamer-based delivery system are also briefly elaborated.
Mathematical approaches in estimating aptamer-target binding affinity

Thevendran R, Navien TN, Meng X, Wen K, Lin Q, Sarah S, et al.

Anal Biochem, 2020 07 01;600:113742.
PMID: 32315616 DOI: 10.1016/j.ab.2020.113742

The performance of aptamers as versatile tools in numerous analytical applications is critically dependent on their high target binding specificity and selectivity. However, only the technical or methodological aspects of measuring aptamer-target binding affinities are focused, ignoring the equally important mathematical components that play pivotal roles in affinity measurements. In this study, we aim to provide a comprehensive review regarding the utilization of different mathematical models and equations, along with a detailed description of the computational steps involved in mathematically deriving the binding affinity of aptamers against their specific target molecules. Mathematical models ranging from one-site binding to multiple aptameric binding site-based models are explained in detail. Models applied in several different approaches of affinity measurements such as thermodynamics and kinetic analysis, including cooperativity and competitive-assay based mathematical models have been elaborately discussed. Mathematical models incorporating factors that could potentially affect affinity measurements are also further scrutinized.
Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction method combined with the SYBR Green real-time PCR assay

Tan LL, Ahmed SA, Ng SK, Citartan M, Raabe CA, Rozhdestvensky TS, et al.

Food Chem, 2020 Mar 30;309:125654.
PMID: 31678669 DOI: 10.1016/j.foodchem.2019.125654

A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.
Aptamers as the chaperones (Aptachaperones) of drugs-from siRNAs to DNA nanorobots

Citartan M, Kaur H, Presela R, Tang TH

Int J Pharm, 2019 Aug 15;567:118483.
PMID: 31260780 DOI: 10.1016/j.ijpharm.2019.118483

Aptamers, nucleic acid ligands that are specific against their corresponding targets are increasingly employed in a variety of applications including diagnostics and therapeutics. The specificity of the aptamers against their targets is also used as the basis for the formulation of the aptamer-based drug delivery system. In this review, we aim to provide an overview on the chaperoning roles of aptamers in acting as the cargo or load carriers, delivering contents to the targeted sites via cell surface receptors. Internalization of the aptamer-biomolecule conjugates via receptor-mediated endocytosis and the strategies to augment the rate of endocytosis are underscored. The cargos chaperoned by aptamers, ranging from siRNAs to DNA origami are illuminated. Possible impediments to the aptamer-based drug deliveries such as susceptibility to nuclease resistance, potentiality for immunogenicity activation, tumor heterogeneity are speculated and the corresponding amendment strategies to address these shortcomings are discussed. We prophesy that the future of the aptamer-based drug delivery will take a trajectory towards DNA nanorobot-based assay.

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