Described herein are synthetic methods for producing sequence-specific RNA oligonucleotides that eliminate impurities produced in prior art methods. In one aspect, an end-protected capture DNA complementary to a portion of the product RNA is employed. In another aspect, the template DNA is covalently or noncovalently linked to the RNA polymerase, either directly or through the use of a nontemplate DNA. In a third aspect, a flow chamber is employed. All of the methods can be used in combination.

Described herein are synthetic methods for producing sequence-specific RNA oligonucleotides that eliminate impurities produced in prior art methods. In one aspect, an end-protected capture DNA complementary to a portion of the product RNA is employed. In another aspect, the template DNA is covalently or noncovalently linked to the RNA polymerase, either directly or through the use of a nontemplate DNA. In a third aspect, a flow chamber is employed. All of the methods can be used in combination.

Provided herein are nanostructure-based sequencing methods and systems.

Provided herein are nanostructure-based sequencing methods and systems.

Methods and compositions for in situ detection of circular RNA in a tissue sample are provided.

Methods and compositions for in situ detection of circular RNA in a tissue sample are provided.

Provided herein is a reverse transcriptase mixture comprising a reverse transcriptase and a colored dye at a concentration in the range of 0.003%-1% (v/w). The colored dye may be visually observed during transfer of the mix from one vessel to another and addition of the mix to another mix can be confirmed by eye by observing the colored dye.

This specification discloses a novel methodology for labelling RNA via enzymatic incorporation of a minimally perturbing fluorescent tricyclic cytosine analogue. This analogue is shown to be 100% incorporated in example transcripts and is fully compatible with both in vitro and in cell transcription. Spectroscopic characterization shows that the incorporation rate of the cytosine analogue is on par with its natural counterpart. Using live cell imaging and flow cytometry, labelled mRNAs are efficiently and correctly translated upon transfection into living cells and cell-free systems. The spectral properties of the modified transcripts and their correct translation product allow for their straightforward and simultaneous visualization. This technology therefore offers a general route to understanding the biological behaviour of RNA of interest, including RNA based drugs. The fluorescent tricyclic cytosine analogue has formula (I):

##STR00001##

This specification discloses a novel methodology for labelling RNA via enzymatic incorporation of a minimally perturbing fluorescent tricyclic cytosine analogue. This analogue is shown to be 100% incorporated in example transcripts and is fully compatible with both in vitro and in cell transcription. Spectroscopic characterization shows that the incorporation rate of the cytosine analogue is on par with its natural counterpart. Using live cell imaging and flow cytometry, labelled mRNAs are efficiently and correctly translated upon transfection into living cells and cell-free systems. The spectral properties of the modified transcripts and their correct translation product allow for their straightforward and simultaneous visualization. This technology therefore offers a general route to understanding the biological behaviour of RNA of interest, including RNA based drugs. The fluorescent tricyclic cytosine analogue has formula (I):

##STR00001##

This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an 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 also 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-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), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.