The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).

The development and success of RNA-based vaccines targeting SARS-CoV-2 has awakened new interest in utilizing RNA vaccines against cancer, particularly in the emerging use of self-replicating RNA (srRNA) viral vaccine platforms. These vaccines are based on different single-stranded RNA viruses, which encode RNA for target antigens in addition to replication genes that are capable of massively amplifying RNA messages after infection. The encoded replicase genes also stimulate innate immunity, making srRNA vectors ideal candidates for anti-tumor vaccination. In this review, we summarize different types of srRNA platforms that have emerged and review evidence for their efficacy in provoking anti-tumor immunity to different antigens. These srRNA platforms encompass the use of naked RNA, DNA-launched replicons, viral replicon particles (VRP), and most recently, synthetic srRNA replicon particles. Across these platforms, studies have demonstrated srRNA vaccine platforms to be potent inducers of anti-tumor immunity, which can be enhanced by homologous vaccine boosting and combining with chemotherapies, radiation, and immune checkpoint inhibition. As such, while this remains an active area of research, the past and present trajectory of srRNA vaccine development suggests immense potential for this platform in producing effective cancer vaccines.

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Objectives: The HIV-1 nef gene terminates in a 3’-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. It has been proposed that antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, through translation of in-frame UGA stop codons as selenocysteine (Sec). Here, our aim was to assess whether readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, and whether selenium-based mechanisms might be involved. Material and Methods: To assess UGA codon readthrough, we used fluorescence microscopy image analysis and flow cytometry of HEK 293 cells transfected with full length HIV-1 nef gene expression constructs including the 3’-UGA stop codon and a predicted thioredoxin reductase 1 (TXNRD1) antisense region spanning the UGA codon, engineered with a downstream in-frame green fluorescent protein (GFP) reporter gene. These were designed so that GFP can only be expressed by translational recoding of the UGA codon, that is, if the UGA codon is translated as an amino acid or bypassed by ribosomal hopping. To assess readthrough efficiency, appropriate mutant control constructs were used for 100% and 0% readthrough. We used anti-TXNRD1 siRNA to assess the possible role of the proposed antisense interaction in this event, by knockdown of TXNRD1 mRNA levels. Results: UGA stop codon readthrough efficiency for the wild-type nef construct was estimated by flow cytometry to be about 19% (P < 0.0001). siRNA knockdown of TXNRD1 mRNA resulted in a 67% decrease in GFP expression in this system relative to control cells (P < 0.0001), presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough (i.e. the TXNRD1 SECIS element). Addition of 20 nM sodium selenite to the media enhanced stop codon readthrough in the pNefATI1 plasmid construct by >100% (P < 0.0001), that is, more than doubled the amount of readthrough product, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism. Conclusion: Our results show that readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, that this is dependent on selenium concentration, and the presence of TXNRD1 mRNA, supporting the proposed antisense tethering interaction.

The HIV-1 nef gene terminates in a 3&rsquo;-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. Antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element (Taylor et al, 2016) [1]. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine (SeC). Here we show that readthrough of the 3&rsquo;-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells. This was accomplished via fluorescence microscopy image analysis and flow cytometry of HEK 293 cells, transfected with engineered GFP reporter gene plasmid constructs, in which GFP can only be expressed by translational recoding of the UGA codon. SiRNA knockdown of thioredoxin reductase 1 (TR1) mRNA resulted in a 67% decrease in GFP expression, presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough, thus supporting the proposed ATI. Addition of 20 nM sodium selenite to the media significantly enhanced stop codon readthrough in the pNefATI1 plasmid construct, by &gt;100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.