Cleavable primers are incorporated into single cell analysis workflows to reduce and/or eliminate misprimed nucleic acid amplicons. Specifically, cleavable primers can introduce restriction endonuclease cleavage sites into misprimed nucleic acid amplicons. For example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising DNA misprimed by an RNA primer. As another example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising cDNA misprimed by a DNA primer. Such amplicons can then be cleaved by a restriction endonuclease to remove them from identification and association in subsequent nucleic acid sequencing.

Cleavable primers are incorporated into single cell analysis workflows to reduce and/or eliminate misprimed nucleic acid amplicons. Specifically, cleavable primers can introduce restriction endonuclease cleavage sites into misprimed nucleic acid amplicons. For example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising DNA misprimed by an RNA primer. As another example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising cDNA misprimed by a DNA primer. Such amplicons can then be cleaved by a restriction endonuclease to remove them from identification and association in subsequent nucleic acid sequencing.

This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, lncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, lncRNA's splice-site variants, translocations, and miRNA.

The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, lncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, lncRNA's splice-site variants, translocations, and miRNA.

The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for detecting a region of interest in a target nucleic acid, wherein hybridization between an interrogatory region of a probe and a region of interest of the target nucleic acid is blocked by a blocking strand unless the interrogatory region is complementary to the region of interest. In some aspects, the methods provided herein increase specificity of detecting a region of interest in a target nucleic acid (e.g., a SNP in an RNA molecule). In some aspects, the presence, amount, and/or identity of a region of interest in a target nucleic acid is analyzed in situ. Also provided are polynucleotides, sets of polynucleotides, compositions, and kits for use in accordance with the methods, for example for RNA-targeting padlock probe-mediated SNP detection.

The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for detecting a region of interest in a target nucleic acid, wherein hybridization between an interrogatory region of a probe and a region of interest of the target nucleic acid is blocked by a blocking strand unless the interrogatory region is complementary to the region of interest. In some aspects, the methods provided herein increase specificity of detecting a region of interest in a target nucleic acid (e.g., a SNP in an RNA molecule). In some aspects, the presence, amount, and/or identity of a region of interest in a target nucleic acid is analyzed in situ. Also provided are polynucleotides, sets of polynucleotides, compositions, and kits for use in accordance with the methods, for example for RNA-targeting padlock probe-mediated SNP detection.

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.