We have used a DNA-aptamer tethered to an atomic force microscope probe to carry out recognition imaging of IgE molecules attached to a mica substrate. The recognition was efficient (approximately 90%) and specific, being blocked by injection of IgE molecules in solution, and not being interfered with by high concentrations of a second protein. The signal/noise ratio of the recognition signal was better than that obtained with antibodies, despite the fact that the average force required to break the aptamer-protein bonds was somewhat smaller.

We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a K d of 21 nM, and discriminates against both the non-acetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody, but shows significantly greater specificity (15-fold versus 2,400-fold) for the target molecule. This result demonstrates that aptamers that are both modification and location specific can be generated to bind specific protein post-translational modifications. KeywordsAptamers; post-translational modifications; antibodies; histone proteins The development of high quality affinity reagents to human proteins represents a major challenge in basic and applied biomedicine. Many large-scale biological assays rely on the use of antibodies to interrogate the nature and function of the human proteome. 1 Unfortunately, only a small portion of human proteins have antibodies that are available for use in routine molecular and cellular biology assays. 2 Even less common are antibodies with high affinity and specificity to specific post-translational modifications (PTMs) and caution is often urged when using antibodies to detect modified proteins in biological samples. 2,3 This shortfall has *To whom correspondence should be addressed. Telephone: (480) 727-0392, email: E-mail: john.chaput@asu.edu. Aptamers, pieces of single-stranded DNA or RNA that fold into three-dimensional structures with binding sites that are complementary in shape and charge to target antigens, have received much attention as possible alternatives to traditional antibodies. 5 Because these molecules can be produced in vitro by test-tube evolution methods, their recognition and binding properties can be tailored to specific target antigens. Indeed, aptamers have now been created to bind virtually any target including ions, small molecules, drugs, peptides, proteins, and even whole cells. 6 Despite these advances, very few aptamers have been identified that bind specific protein PTMs. In fact, only one literature-reported aptamer exists that binds a PTM and this aptamer shows only a 10-fold preference against the unmodified target. 7,8 In this report, we address the question of whether aptamers can be created that bind subtle PTMs and distinguish their site of occurrence in a protein sequence. We chose histone H4 acetylated at lysine 16 (H4-K16Ac) as our target due to the importance of this modification in regulating gene activation and silencing. 9 Because the K4-H16Ac modification is located on the N-terminal tail, which is a region of the protein that remains unfolded and accessible to chromatin modifying enzymes when assembled into nucleosomes, we used a 15-mer peptide containing residues Gly6 to Lys20 to represent this portion of the protein. 10 The use of synthetic...

BackgroundThe emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed.MethodsTargeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively.ResultsThe limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples.ConclusionThis work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Recognition imaging microscopy is an analytical technique used to map the topography and chemical identity of specific protein molecules present in complex biological samples. The technique relies on the use of antibodies tethered to the cantilever tip of an AFM probe to detect cognate antigens deposited onto a mica surface. Despite the power of this technique to resolve single molecules with nanometer-scale spacing, the recognition step remains limited by the availability of suitable quality Supporting Information Available: Detailed protocols, sequences, mFold structures, and AFM images are available in the supplementary section. This material is available free of charge via the Internet at http://pubs.acs.org. antibodies. Here we report the in vitro selection and recognition imaging of anti-histone H4 aptamers. In addition to identifying aptamers to highly basic proteins, these results suggest that aptamers provide an efficient, cost effective route to highly selective affinity reagents for recognition imaging microscopy. NIH Public AccessRecognition imaging microscopy is an analytical technique used to map the topography and chemical identity of specific protein molecules present in complex biological samples. 1-3 The technique relies on the use of affinity reagents immobilized to the cantilever tip of an atomic force microscope (AFM) to identify the precise location of a single protein in an aqueous environment. Since surface images can be acquired in near real-time, recognition imaging has been used to study many time dependent processes. 1 Despite the ability of this technique to resolve single molecules with nanometer-scale spacing, the recognition step remains limited by the availability of antibodies of suitable quality. In our work on chromatin remodeling we have found that many commercial antibodies developed to recognize DNA binding proteins show batch-to-batch variation in performance and mild to severe cross-reactivity with other proteins. 4 To overcome these limitations we initiated an investigation into alternative affinity reagents as antibody mimics in recognition imaging microscopy. 5 Here we report the in vitro selection and evaluation of DNA aptamers selected to bind histone H4 tails.Aptamers are nucleic acid molecules that exhibit antibody-like properties by adopting structures that are complementary in shape and charge to a selected target. 6 In contrast to antibodies, aptamers are smaller in size, easier to engineer, and can be generated relatively quickly using standard in vitro selection technologies. Although aptamers have been selected to bind a diverse array of targets with high affinity and specificity, 6 some concern remains over their ability to bind highly charged molecules due to the potential for nonspecific binding. 7 Recognizing that histones are highly charged proteins that contain many lysine and arginine residues, we wondered whether aptamers could be selected to recognize different histone classes.To address this question we used an in vitro selection protocol th...

Background: Plasmodium falciparum circumsporozoite protein (PfCSP) is a potential malaria vaccine candidate, but various polymorphisms of the pfcsp gene among global P. falciparum population become the major barrier to the effectiveness of vaccines. This study aimed to investigate the genetic polymorphisms and natural selection of pfcsp in Bioko and the comparison among global P. falciparum population. Methods: From January 2011 to December 2018, 148 blood samples were collected from P. falciparum infected Bioko patients and 96 monoclonal sequences of them were successfully acquired and analysed with 2200 global pfcsp sequences mined from MalariaGEN Pf3k Database and NCBI. Results: In Bioko, the N-terminus of pfcsp showed limited genetic variations and the numbers of repetitive sequences (NANP/NVDP) were mainly found as 40 (35%) and 41 (34%) in central region. Most polymorphic characters were found in Th2R/Th3R region, where natural selection (p > 0.05) and recombination occurred. The overall pattern of Bioko pfcsp gene had no obvious deviation from African mainland pfcsp (Fst = 0.00878, p < 0.05). The comparative analysis of Bioko and global pfcsp displayed the various mutation patterns and obvious geographic differentiation among populations from four continents (p < 0.05). The global pfcsp C-terminal sequences were clustered into 138 different haplotypes (H_1 to H_138). Only 3.35% of sequences matched 3D7 strain haplotype (H_1). Conclusions: The genetic polymorphism phenomena of pfcsp were found universal in Bioko and global isolates and the majority mutations located at T cell epitopes. Global genetic polymorphism and geographical characteristics were recommended to be considered for future improvement of malaria vaccine design.