Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization

The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.

That pesky nucleic acid molecule in a protein coat

In March it seemed not only surreal but impossible to comprehend that the coronavirus would ever venture near our shores – and yet here it is. Jane Hanley looks at the effects of the pandemic on the emotional wellbeing of parents and professionals alike

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

ABSTRACTCRISPR-Cas systems are bacterial immune systems that target invading nucleic acid. The hallmark of CRISPR-Cas systems is the CRISPR array, a genetic locus that includes short sequences known as “spacers”, that are derived from invading nucleic acid. Upon exposure to an invading nucleic acid molecule, bacteria/archaea with functional CRISPR-Cas systems can add new spacers to their CRISPR arrays in a process known as “adaptation”. In type I CRISPR-Cas systems, which represent the majority of CRISPR-Cas systems found in nature, adaptation can occur by two mechanisms: naïve and primed. Here, we show that, for the archetypal type I-E CRISPR-Cas system from Escherichia coli, primed adaptation occurs at least 1,000 times more efficiently than naïve adaptation. By initiating primed adaptation on the E. coli chromosome, we show that spacers can be acquired across distances of >100 kb from the initially targeted site, and we identify multiple factors that influence the efficiency with which sequences are acquired as new spacers. Thus, our data provide insight into the mechanism of primed adaptation.[This paper has been peer reviewed, with Ailong Ke (Cornell University) serving as the editor. Reviews and point-by-point response, and a marked-up version of the edited manuscript are provided as supplementary files.]

Identifying triplex binding rules in vitro leads to creation of a new synthetic regulatory tool in vivo

SummaryNature provides a rich toolbox of dynamic nucleic acid structures that are widespread in cells and affect multiple biological processes1. Recently, non-canonical structures gained renewed scientific and biotechnological interest2,3. One particularly intriguing form of such structures are triplexes4 in which a single-stranded nucleic acid molecule interacts via Hoogsteen bonds with a DNA/RNA double helix5. Despite extensive research in vitro6–9, the underlying rules for triplex formation remain debated and evidence for triplexes in vivo is circumstantial10–12. Here, we demonstrate the development of a deep-sequencing platform termed Triplex-Seq to systematically refine the DNA triplex code and identify high affinity triplex forming oligo (TFO) variants. We identified a preference for short G-rich motifs using an oligo-library with a mix of all four bases. These high-information content motifs formed specific high-affinity triplexes in a pH-independent manner and stability was increased with G-rich double-stranded molecules. We then conjugated one high-affinity and one low-affinity variant to a VP48 peptide and studied these synthetic biomolecules in mammalian cells. Using these peptide-oligo constructs (POCs), we demonstrated possible triplex-induced down-regulation activity in 544 differentially expressed genes. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules, which in turn has led to the development of a functional non-genetically encoded regulatory tool for in vivo applications.

Virological Factors Associated with the Occurrence of HBV Reactivation in Patients with Resolved HBV Infection Analyzed through Ultradeep Sequencing

Background Hepatitis B virus reactivation (HBVr) is an important complication of immunosuppressive drug therapy. It can occur via both virological and host factors; however, the underlying mechanisms remain largely unknown. Methods We examined serum samples derived from patients with HBVr and those with acute hepatitis B (AHB). HBV DNA was amplified the targeted nucleic acid molecule and analyzed by next-generation sequencing. Results The percentage of patients infected with genotype Bj among the HBVr patients was significantly higher than that in the AHB patients. The frequency of mutation sites in the whole HBV genome, especially in the envelope region, in the HBVr was significantly higher than that in the AHB. The prevalence of the S3N amino acid substitution in the envelope protein and mutations at positions G1896A and G1899A in the precore region were significantly higher in the HBVr compared to AHB. The population of S3N aa substitution and nt G1896A and G1899A mutations in each individual showed quite a similar percentage of occurrence. Conclusions We identified specific virological factors in patients with HBVr through ultra-deep sequencing. Our findings would be beneficial for the elucidation of mechanisms underlying HBVr development and for disease control.

The Metric Structure of Nucleic Acids and the Higher Dimension of Their Constituents

The purpose of the study is to build a metric model of the structure of a nucleic acid molecule, taking into account the higher dimension of its components, the actual lengths of chemical bonds, and the angles between them. Calculations by the constructed model showed the closeness of the known experimental characteristic parameters of the nucleic acid (helix diameter, period length) and the calculated values of these parameters. The internal degree of freedom of the nucleic acid molecule is revealed: the angle of rotation of the phosphoric acid residue relative to the chemical bond connecting it to the five-carbon sugar molecule. It is established that the value of this angle determines the shape of the nucleic acid molecule. It is claimed that the process of transmission of hereditary information and protein synthesis occurs in a space of high dimensionality in ribosomes.

Genomic DNA preparation enabling multiple replicate reads for accurate nanopore sequencing

Sequencing at single-nucleotide resolution using nanopore devices is performed with reported error rates 10.5-20.7% (Ip et al., 2015). Since errors occur randomly during sequencing, repeating the sequencing procedure for the same DNA strands several times can generate sequencing results based on consensus derived from replicate readings, thus reducing overall error rates. The method presented in this manuscript constructs copies of a nucleic acid molecule that are consecutively connected to the nucleic acid molecule. Such copies are useful because they can be sequenced by a nanopore device, enabling replicate reads, thus improving overall sequencing accuracy.