Compositions and methods for rapid rna-adenylation and rna sequencing
AssigneeCornell University
InventorsShu-Bing QIAN, Leiming DONG, Xin SHU
Life of this patent
Estimating the term…
Abstract
Provided are compositions, methods, kits, and articles of manufacture for use in RNA sequencing. The approach is referred to as easy RNA-adenylation sequencing (“Ezra-seq”). The approach provides an alternative to 3′ end linker ligation and circularization by way of an enzymatic system capable of 3′ end poly(A) tailing and 5′-end adenylation for the same RNA, using two separate enzymes, or a single fusion protein. The two enzymes or the fusion protein containing them as distinct segments are a cyclase and a polymerase. The method allows for single container processing of RNA into cDNA,
Claims14
A method for determining nucleotide sequences of RNA polynucleotides, the method comprising:
- a) providing a plurality of RNA fragments obtained from the RNA polynucleotides;
- b) enzymatically phosphorylating 5′ ends of the plurality of RNA fragments to provide a plurality of RNA fragments comprising mono-phosphorylated 5′ ends;
- c) enzymatically dephosphorylating 3′ ends of the plurality of RNA fragments to provide a plurality of RNA fragments comprising free 3′ hydroxyls;
- d) enzymatically adenylylating phosphorylated 5′ ends of the plurality of RNA fragments to provide a plurality of 5′ mono-adenylated RNA fragments;
- e) enzymatically polyadenylating 3′ ends of the plurality of RNA fragments comprising the free 3′ hydroxyls to provide a plurality of RNA fragments comprising polyadenylated 3′ ends;
- f) ligating oligonucleotide adapters to the 5′ ends of the plurality of 5′ mono-adenylated RNA fragments, the oligonucleotide adapters optionally comprising a DNA/RNA hybrid 3′ end, the DNA/RNA hybrid 3′ end optionally comprising rCrC-OH or rGrG-OH as the RNA component of the DNA/RNA hybrid, to provide a plurality of RNA fragments comprising the oligonucleotide adapters at the 5′ ends;
- g) generating cDNAs from the plurality of RNA fragments of f);
- h) amplifying the cDNAs;
- and
- i) determining nucleotide sequences of the cDNAs, thereby determining the nucleotide sequences of the RNA polynucleotides.
-17. (canceled)
An article of manufacture comprising at least one sealed container, the at least one sealed container containing at least a mixture of two distinct proteins or the fusion protein for use in RNA sequencing, the two distinct proteins or the fusion protein comprising a poly(A) polymerase and an RNA 3′-phosphate cyclase, the article of manufacture further comprising printed material that provides an indication that contents of the article of manufacture are for use in RNA sequencing.
Dependencies are inferred from the claim text.