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1.
Anal Chem ; 94(21): 7619-7627, 2022 May 31.
Article in English | MEDLINE | ID: covidwho-1852361

ABSTRACT

The COVID-19 pandemic has revealed how an emerging pathogen can cause a sudden and dramatic increase in demand for viral testing. Testing pooled samples could meet this demand; however, the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR), the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce detection of intact virus by exogenous-nucleotide reaction (DIVER), a method that quantifies intact virus and is robust to sample dilution. As demonstrated using two models of severe acute respiratory syndrome coronavirus 2, DIVER first tags membraned particles with exogenous oligonucleotides, then captures the tagged particles on beads functionalized with a virus-specific capture agent (in this instance, angiotensin-converting enzyme 2), and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and spike-pseudotyped lentivirus, we show that DIVER can detect 1 × 105 liposomes and 100 plaque-forming units of lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well positioned for efficient sample pooling and clinical validation.


Subject(s)
COVID-19 , Pandemics , COVID-19/diagnosis , Humans , Liposomes , Oligonucleotides , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
2.
Viruses ; 14(4)2022 03 25.
Article in English | MEDLINE | ID: covidwho-1834920

ABSTRACT

In only two years, the coronavirus disease 2019 (COVID-19) pandemic has had a devastating effect on public health all over the world and caused irreparable economic damage across all countries. Due to the limited therapeutic management of COVID-19 and the lack of tailor-made antiviral agents, finding new methods to combat this viral illness is now a priority. Herein, we report on a specific oligonucleotide-based RNA inhibitor targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It displayed remarkable spontaneous cellular uptake, >94% efficiency in reducing RNA-dependent RNA polymerase (RdRp) RNA levels in transfected lung cell lines, and >98% efficiency in reducing SARS-CoV-2 RNA levels in samples from patients hospitalized with COVID-19 following a single application.


Subject(s)
COVID-19 , Oligonucleotides , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Humans , Oligonucleotides/pharmacology , Oligonucleotides/therapeutic use , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics
3.
AAPS PharmSciTech ; 23(5): 135, 2022 May 09.
Article in English | MEDLINE | ID: covidwho-1833435

ABSTRACT

Lipid nanoparticles (LNPs) can be used as delivery vehicles for nucleic acid biotherapeutics. In fact, LNPs are currently being used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. Cationic LNPs composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) LNPs have been classified as one of the most efficient gene delivery systems and are being tested in numerous clinical trials. The objective of this study was to examine the effect of the molar ratio of DOTAP/chol, PEGylation, and lipid to mRNA ratio on mRNA transfection, and explore the applications of DOTAP/chol LNPs in pDNA and oligonucleotide transfection. Here we showed that PEGylation significantly decreased mRNA transfection efficiency of DOTAP/chol LNPs. Among non-PEGylated LNP formulations, 1:3 molar ratio of DOTAP/chol in DOTAP/chol LNPs showed the highest mRNA transfection efficiency. Furthermore, the optimal ratio of DOTAP/chol LNPs to mRNA was tested to be 62.5 µM lipid to 1 µg mRNA. More importantly, these mRNA-loaded nanoparticles were stable for 60 days at 4 °C storage without showing reduction in transfection efficacy. We further found that DOTAP/chol LNPs were able to transfect pDNA and oligonucleotides, demonstrating the ability of these LNPs to transport the cargo into the cell nucleus. The influence of various factors in the formulation of DOTAP/chol cationic LNPs is thus described and will help improve drug delivery of nucleic acid-based vaccines and therapies.


Subject(s)
COVID-19 , Nanoparticles , COVID-19 Vaccines , Cations , Cholesterol , Fatty Acids, Monounsaturated , Humans , Liposomes , Oligonucleotides , Propane , Quaternary Ammonium Compounds , RNA, Messenger/genetics
5.
Biochem Biophys Res Commun ; 614: 207-212, 2022 Jul 23.
Article in English | MEDLINE | ID: covidwho-1814155

ABSTRACT

Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , DNA/metabolism , Humans , Oligonucleotides/metabolism , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
6.
Anal Chem ; 94(18): 6760-6770, 2022 05 10.
Article in English | MEDLINE | ID: covidwho-1805541

ABSTRACT

The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , DNA , Digestion , Humans , Nucleic Acid Amplification Techniques , Oligonucleotides , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
7.
Int J Mol Sci ; 23(8)2022 Apr 14.
Article in English | MEDLINE | ID: covidwho-1792661

ABSTRACT

The recent development of mRNA vaccines against the SARS-CoV-2 infection has turned the spotlight on the potential of nucleic acids as innovative prophylactic agents and as diagnostic and therapeutic tools. Until now, their use has been severely limited by their reduced half-life in the biological environment and the difficulties related to their transport to target cells. These limiting aspects can now be overcome by resorting to chemical modifications in the drug and using appropriate nanocarriers, respectively. Oligonucleotides can interact with complementary sequences of nucleic acid targets, forming stable complexes and determining their loss of function. An alternative strategy uses nucleic acid aptamers that, like the antibodies, bind to specific proteins to modulate their activity. In this review, the authors will examine the recent literature on nucleic acids-based strategies in the COVID-19 era, focusing the attention on their applications for the prophylaxis of COVID-19, but also on antisense- and aptamer-based strategies directed to the diagnosis and therapy of the coronavirus pandemic.


Subject(s)
COVID-19 , Nucleic Acids , Humans , Nanomedicine , Nucleic Acids/therapeutic use , Oligonucleotides/chemistry , Oligonucleotides/therapeutic use , SARS-CoV-2
8.
Mikrochim Acta ; 189(4): 171, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1777732

ABSTRACT

Gold nanotriangles (AuNTs) functionalized with dithiolated oligonucleotides have been employed to develop an amplification-free electrochemical biosensor for SARS-CoV-2 in patient samples. Gold nanotriangles, prepared through a seed-mediated growth method and exhaustively characterized by different techniques, serve as an improved electrochemical platform and for DNA probe immobilization. Azure A is used as an electrochemical indicator of the hybridization event. The biosensor detects either single stranded DNA or RNA sequences of SARS-CoV-2 of different lengths, with a low detection limit of 22.2 fM. In addition, it allows to detect point mutations in SARS-CoV-2 genome with the aim to detect more infective SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron. Results obtained with the biosensor in nasopharyngeal swab samples from COVID-19 patients show the possibility to clearly discriminate between non-infected and infected patient samples as well as patient samples with different viral load. Furthermore, the results correlate well with those obtained by the gold standard technique RT-qPCR, with the advantage of avoiding the amplification process and the need of sophisticated equipment.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Nucleic Acid Hybridization , Oligonucleotides , SARS-CoV-2/genetics
9.
Pharmacol Ther ; 230: 107967, 2022 02.
Article in English | MEDLINE | ID: covidwho-1757736

ABSTRACT

The concepts of developing RNAs as new molecular entities for therapies have arisen again and again since the discoveries of antisense RNAs, direct RNA-protein interactions, functional noncoding RNAs, and RNA-directed gene editing. The feasibility was demonstrated with the development and utilization of synthetic RNA agents to selectively control target gene expression, modulate protein functions or alter the genome to manage diseases. Rather, RNAs are labile to degradation and cannot cross cell membrane barriers, making it hard to develop RNA medications. With the development of viable RNA technologies, such as chemistry and pharmaceutics, eight antisense oligonucleotides (ASOs) (fomivirsen, mipomersen, eteplirsen, nusinersen, inotersen, golodirsen, viltolarsen and casimersen), one aptamer (pegaptanib), and three small interfering RNAs (siRNAs) (patisiran, givosiran and lumasiran) have been approved by the United States Food and Drug Administration (FDA) for therapies, and two mRNA vaccines (BNT162b2 and mRNA-1273) under Emergency Use Authorization for the prevention of COVID-19. Therefore, RNAs have become a great addition to small molecules, proteins/antibodies, and cell-based modalities to improve the public health. In this article, we first summarize the general characteristics of therapeutic RNA agents, including chemistry, common delivery strategies, mechanisms of actions, and safety. By overviewing individual RNA medications and vaccines approved by the FDA and some agents under development, we illustrate the unique compositions and pharmacological actions of RNA products. A new era of RNA research and development will likely lead to commercialization of more RNA agents for medical use, expanding the range of therapeutic targets and increasing the diversity of molecular modalities.


Subject(s)
COVID-19 , Humans , Oligonucleotides , RNA, Small Interfering , SARS-CoV-2 , United States , Vaccination
10.
Ann Clin Transl Neurol ; 9(3): 404-409, 2022 03.
Article in English | MEDLINE | ID: covidwho-1750287

ABSTRACT

The study reports real world data in type 2 and 3 SMA patients treated for at least 2 years with nusinersen. Increase in motor function was observed after 12 months and during the second year. The magnitude of change was variable across age and functional subgroup, with the largest changes observed in young patients with higher function at baseline. When compared to natural history data, the difference between study cohort and untreated patients swas significant on both Hammersmith Functional Motor Scale and Revised Upper Limb Module both at 12 months and at 24 months.


Subject(s)
Muscular Atrophy, Spinal , Cohort Studies , Humans , Muscular Atrophy, Spinal/drug therapy , Oligonucleotides/pharmacology , Oligonucleotides/therapeutic use , Upper Extremity
11.
Nanoscale ; 14(13): 5112-5120, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1747170

ABSTRACT

The early stages of the COVID-19 pandemic punctuated the need for rapid, mass testing for early detection of viral infection. Carbon dots are easily synthesized, cost-effective fluorescent nanoparticles whose surface functionalities enable facile conjugation with biorecognition elements suitable for  molecular detection of viral RNA. Herein, we report that a pair of complementary antisense oligonucleotide (ASO) sequences can lead to a highly specific molecular aggregation of dual colour carbon dots (CDs) in the presence of SARS-CoV-2 RNA. The nanoprobes used ASOs highly specific to the N-gene of SARS-COV-2. When the ASOs are conjugated to blue and yellow citric acid-derived CDs, the combination of the ASO-CD pairs facilitates aggregation-induced emission enhancement (AIEE) of the measured fluorescence after hybridization with SARS-CoV-2 RNA. We found the sensor capable of differentiating between MERS-CoV and SARS-CoV-2 samples and was found to have a limit of detection of 81 copies per µL. Additionally, we used dialysis to demonstrate that the change in emission upon aggregation is dependent on the compositional heterogeneity of the conjugated-carbon dot mixture.


Subject(s)
COVID-19 , RNA, Viral , COVID-19/diagnosis , Carbon , Color , Humans , Oligonucleotides , Oligonucleotides, Antisense , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics
12.
Wiley Interdiscip Rev Nanomed Nanobiotechnol ; 14(3): e1785, 2022 05.
Article in English | MEDLINE | ID: covidwho-1718500

ABSTRACT

The emergence of SARS-COV-2, the causative agent of new coronavirus disease (COVID-19) has become a pandemic threat. Early and precise detection of the virus is vital for effective diagnosis and treatment. Various testing kits and assays, including nucleic acid detection methods, antigen tests, serological tests, and enzyme-linked immunosorbent assay (ELISA), have been implemented or are being explored to detect the virus and/or characterize cellular and antibody responses to the infection. However, these approaches have inherent drawbacks such as nonspecificity, high cost, are characterized by long turnaround times for test results, and can be labor-intensive. Also, the circulating SARS-COV-2 variant of concerns, reduced antibody sensitivity and/or neutralization, and possible antibody-dependent enhancement (ADE) have warranted the search for alternative potent therapeutics. Aptamers, which are single-stranded oligonucleotides, generated artificially by SELEX (Evolution of Ligands by Exponential Enrichment) may offer the capacity to generate high-affinity neutralizers and/or bioprobes for monitoring relevant SARS-COV-2 and COVID-19 biomarkers. This article reviews and discusses the prospects of implementing aptamers for rapid point-of-care detection and treatment of SARS-COV-2. We highlight other SARS-COV-2 targets (N protein, spike protein stem-helix), SELEX augmented with competition assays and in silico technologies for rapid discovery and isolation of theranostic aptamers against COVID-19 and future pandemics. It further provides an overview on site-specific bioconjugation approaches, customizable molecular scaffolding strategies, and nanotechnology platforms to engineer these aptamers into ultrapotent blockers, multivalent therapeutics, and vaccines to boost both humoral and cellular immunity against the virus. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > Biosensing Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Oligonucleotides , Pandemics/prevention & control , Theranostic Nanomedicine
13.
Nucleic Acids Res ; 50(1): 333-349, 2022 01 11.
Article in English | MEDLINE | ID: covidwho-1591186

ABSTRACT

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.


Subject(s)
COVID-19/virology , Lung/virology , RNA, Small Interfering , RNA, Viral , SARS-CoV-2/genetics , Virus Replication , 3' Untranslated Regions , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cell Survival , Databases, Genetic , HEK293 Cells , Humans , Nucleic Acid Conformation , Oligonucleotides , Open Reading Frames , RNA, Small Interfering/metabolism
14.
Molecules ; 26(22)2021 Nov 22.
Article in English | MEDLINE | ID: covidwho-1534202

ABSTRACT

The 5',8-cyclo-2'-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered-the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5'-end side of cdPu than on its 3'-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.


Subject(s)
DNA Damage , DNA Repair , DNA, Mitochondrial/metabolism , Oligonucleotides , Purine Nucleosides , Animals , CHO Cells , Cricetulus , Oligonucleotides/chemistry , Oligonucleotides/pharmacology , Purine Nucleosides/chemistry , Purine Nucleosides/pharmacology
15.
PLoS One ; 16(11): e0260087, 2021.
Article in English | MEDLINE | ID: covidwho-1528723

ABSTRACT

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Real-Time Polymerase Chain Reaction/methods , Adult , COVID-19 Nucleic Acid Testing/standards , Female , Humans , Male , Nucleic Acid Denaturation , Oligonucleotides/chemistry , Real-Time Polymerase Chain Reaction/standards , Respiratory Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Saliva/virology , Sensitivity and Specificity
16.
Anal Methods ; 13(45): 5400-5417, 2021 11 25.
Article in English | MEDLINE | ID: covidwho-1506380

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel infectious member of the coronavirus family, has caused millions of cases of infection and deaths all over the world, and been declared a pandemic by the World Health Organization. Conventional laboratory-based diagnostic testing has faced extreme difficulties in meeting the overwhelming demand for testing worldwide, and this has brought about a pressing need for cost-effective rapid diagnosis. There has been a surge in the number of prototypes of diagnostic kits developed, although many of these have been found to be lacking in terms of their accuracy and sensitivity. One type of chip-based diagnostic platform is the aptamer-based biosensor. Aptamers are artificially synthesized oligonucleotides that are capable of specifically binding to a target antigen. As of now, some aptamers have been reported for SARS-CoV-2. Although many ultrasensitive aptasensors have been developed for viruses, few have been successfully adapted for SARS-CoV-2 detection. Our review discusses the recent developments in the domain of SARS-CoV-2 specific aptamer isolation, the design of electrochemical and optical aptasensors, and the implications of aptasensor-based COVID-19 diagnosis.


Subject(s)
Biosensing Techniques , COVID-19 , COVID-19 Testing , Humans , Oligonucleotides , SARS-CoV-2
17.
Adv Sci (Weinh) ; 8(23): e2101166, 2021 12.
Article in English | MEDLINE | ID: covidwho-1473797

ABSTRACT

Lipid-based nanoparticles have been applied extensively in drug delivery and vaccine strategies and are finding diverse applications in the coronavirus disease 2019 (COVID-19) pandemic-from vaccine-component encapsulation to modeling the virus, itself. High-throughput, highly flexible methods for characterization are of great benefit to the development of liposomes featuring surface proteins. DNA-directed patterning is one such method that offers versatility in immobilizing and segregating lipid-based nanoparticles for subsequent analysis. Here, oligonucleotides are selectively conjugated onto a glass substrate and then hybridized to complementary oligonucleotides tagged to liposomes, patterning them with great control and precision. The power of DNA-directed patterning is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-S-liposomes-that presents the SARS-CoV-2 spike (S) protein on its surface. Patterning a mixture of S-liposomes and liposomes that display the tetraspanin CD63 to discrete regions of a substrate shows that angiotensin-converting enzyme 2 (ACE2) specifically binds to S-liposomes. Subsequent introduction of S-liposomes to ACE2-expressing cells tests the biological function of S-liposomes and shows agreement with DNA-directed patterning-based assays. Finally, multiplexed patterning of S-liposomes verifies the performance of commercially available neutralizing antibodies against the two S variants. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/diagnosis , Liposomes/chemistry , Nanoparticles/chemistry , Oligonucleotides/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , COVID-19/virology , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Liposomes/metabolism , Microscopy, Confocal , Oligonucleotides/metabolism , Protein Binding , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Tetraspanins/chemistry , Tetraspanins/metabolism
18.
Talanta ; 236: 122841, 2022 Jan 01.
Article in English | MEDLINE | ID: covidwho-1458899

ABSTRACT

A rapid detection test for SARS-CoV-2 is urgently required to monitor virus spread and containment. Here, we describe a test that uses nanoprobes, which are gold nanoparticles functionalized with an aptamer specific to the spike membrane protein of SARS-CoV-2. An enzyme-linked immunosorbent assay confirms aptamer binding with the spike protein on gold surfaces. Protein recognition occurs by adding a coagulant, where nanoprobes with no bound protein agglomerate while those with sufficient bound protein do not. Using plasmon absorbance spectra, the nanoprobes detect 16 nM and higher concentrations of spike protein in phosphate-buffered saline. The time-varying light absorbance is examined at 540 nm to determine the critical coagulant concentration required to agglomerates the nanoprobes, which depends on the protein concentration. This approach detects 3540 genome copies/µl of inactivated SARS-CoV-2.


Subject(s)
COVID-19 , Metal Nanoparticles , Gold , Humans , Oligonucleotides , SARS-CoV-2
19.
Biomol NMR Assign ; 15(1): 85-89, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384621

ABSTRACT

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b ("X" or macro domain); indeed all of them are folded in a three-layer α/ß/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Magnetic Resonance Spectroscopy , SARS-CoV-2/chemistry , Carbon Isotopes , Drug Design , Hydrogen , Nitrogen Isotopes , Oligonucleotides/chemistry , Protein Domains , Protein Structure, Secondary , Virus Replication
20.
Pharmacol Ther ; 230: 107967, 2022 02.
Article in English | MEDLINE | ID: covidwho-1356395

ABSTRACT

The concepts of developing RNAs as new molecular entities for therapies have arisen again and again since the discoveries of antisense RNAs, direct RNA-protein interactions, functional noncoding RNAs, and RNA-directed gene editing. The feasibility was demonstrated with the development and utilization of synthetic RNA agents to selectively control target gene expression, modulate protein functions or alter the genome to manage diseases. Rather, RNAs are labile to degradation and cannot cross cell membrane barriers, making it hard to develop RNA medications. With the development of viable RNA technologies, such as chemistry and pharmaceutics, eight antisense oligonucleotides (ASOs) (fomivirsen, mipomersen, eteplirsen, nusinersen, inotersen, golodirsen, viltolarsen and casimersen), one aptamer (pegaptanib), and three small interfering RNAs (siRNAs) (patisiran, givosiran and lumasiran) have been approved by the United States Food and Drug Administration (FDA) for therapies, and two mRNA vaccines (BNT162b2 and mRNA-1273) under Emergency Use Authorization for the prevention of COVID-19. Therefore, RNAs have become a great addition to small molecules, proteins/antibodies, and cell-based modalities to improve the public health. In this article, we first summarize the general characteristics of therapeutic RNA agents, including chemistry, common delivery strategies, mechanisms of actions, and safety. By overviewing individual RNA medications and vaccines approved by the FDA and some agents under development, we illustrate the unique compositions and pharmacological actions of RNA products. A new era of RNA research and development will likely lead to commercialization of more RNA agents for medical use, expanding the range of therapeutic targets and increasing the diversity of molecular modalities.


Subject(s)
COVID-19 , Humans , Oligonucleotides , RNA, Small Interfering , SARS-CoV-2 , United States , Vaccination
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