Analysis of mrna 5' capping efficiency
AssigneeCellscript, LLC
InventorsJerome J. Jendrisak, Ronald J. Meis, Daniel Gerhardt, Kari Weber, Gary A. Dahl
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Abstract
The invention relates to methods of quantitating the relative amounts or percentages of RNA molecules that are 5′-capped and that are uncapped in an RNA sample or for quantifying the efficiency of capping of an in vitro-synthesized mRNA of interest that has been subjected to a capping enzyme system. The invention further relates to kits and kit-of-parts for use in said methods and the use of a nuclease that selectively cleaves RNA in a DNA/RNA hybrid in said methods.
Claims21
-17. (canceled)
A method of quantitating the relative amounts or percentages of RNA molecules that are 5′-capped and that are uncapped in an RNA sample or for quantifying the efficiency of capping of an in vitro-synthesized mRNA of interest that has been subjected to a capping enzyme system, said method comprising the following steps a) to e):
- a) providing an RNA sample, wherein said sample comprises:
- i) mRNA with a cap structure at the most 5′-end ribonucleotide of said mRNA of interest;
- and
- ii) mRNA without said cap structure at the most 5′-end ribonucleotide of said mRNA of interest;
- b) contacting said RNA sample with an 5′RNA-DNA-RNA3′ targeting oligonucleotide consisting of a DNA central portion flanked at its 5′-end and its 3′-end by RNA portions, under conditions that permit said targeting oligonucleotide, including said DNA central portion, to hybridize to said mRNA of interest to form a hybrid between the mRNA of interest and the targeting oligonucleotide, wherein said targeting oligonucleotide is further defined as follows:
- i) said DNA central portion comprises or consists of four deoxyribonucleotides in length, wherein said DNA central portion is complementary to a sequence at the 5′-end portion of said mRNA of interest within 15 to 50 bases from said most 5′ end ribonucleotide;
- and
- ii) each of said RNA flanking portions comprises 5 to 15 ribonucleotides in length and said RNA flanking portions are complementary to corresponding RNA portions in said mRNA of interest flanking the sequence hybridized to the targeting oligonucleotide's DNA central portion;
- c) providing a nuclease that selectively cleaves RNA in a DNA/RNA hybrid, resulting in the cleavage of said mRNA of interest in the sequence region hybridized to the targeting oligonucleotide's DNA central portion and the release of:
- i) the cleaved mRNA 5′-end portion fragments with said cap structure;
- and
- ii) the cleaved mRNA 5′-end portion fragments without said cap structure;
- d) electrophoretically separating said released mRNA 5′-end portion fragments;
- and
- e) determining the relative amount of said separated capped fragments and uncapped fragments, thereby quantifying efficiency of mRNA capping in said mRNA of interest.
A method of quantitating the relative amounts or percentages of RNA molecules that are 5′-capped and that are uncapped in a sample or for quantifying the efficiency of mRNA capping of an in vitro-synthesized RNA of interest that has been subjected to a capping enzyme system, said method comprising the following steps a) to e):
- a) providing an RNA sample, wherein said sample comprises:
- i) mRNA with a cap structure at the most 5′-end ribonucleotide of said mRNA of interest;
- and
- ii) mRNA without said cap structure at the most 5′-end ribonucleotide of said mRNA of interest;
- b) contacting said mRNA sample with an 3′RNA-DNA5′ targeting oligonucleotide consisting of a 3′ RNA portion and a 5′ DNA portion, under conditions that permit said targeting oligonucleotide, including said DNA portion, to hybridize to said mRNA of interest to form hybrid between an mRNA of interest and a targeting oligonucleotide, wherein said targeting oligonucleotide is further defined as follows:
- i) said DNA portion comprises or consists of four deoxyribonucleotides in length, wherein said DNA portion is complementary to a sequence at the 5′-end portion of said mRNA of interest within 15 to 50 bases from said most 5′ end ribonucleotide,
- ii) said RNA portion comprises 5 to 15 ribonucleotides in length and said RNA portion is complementary to a corresponding RNA portion in said mRNA of interest flanking the sequence hybridized to the targeting oligonucleotide's DNA portion;
- c) providing a nuclease that selectively cleaves RNA in a DNA/RNA hybrid, resulting in the cleavage of said mRNA of interest in the sequence region hybridized to the targeting oligonucleotide's DNA portion and the release of:
- i) the cleaved mRNA 5′-end portion fragments with said cap structure;
- and
- ii) the cleaved mRNA 5′-end portion fragments without said cap structure;
- d) electrophoretically separating said released mRNA 5′-end portion fragments;
- and
- e) determining the relative amount of said separated capped fragments and uncapped fragments, thereby quantifying efficiency of mRNA capping in said mRNA of interest.
A method of quantitating the relative amounts or percentages of RNA molecules that are 5′-capped and that are uncapped in a sample or for quantifying the efficiency of RNA capping of an in vitro-synthesized RNA of interest that has been subjected to a capping enzyme system, said method comprising the following steps a) to e):
- a) providing an RNA sample, wherein said sample comprises:
- i) mRNA with a cap structure at the most 5′-end ribonucleotide of said mRNA of interest;
- and
- ii) mRNA without said cap structure at the most 5′-end ribonucleotide of said mRNA of interest;
- b) contacting said mRNA sample with a 5′RNA-DNA3″ targeting oligonucleotide consisting of a 5′ RNA portion and 3′ DNA portion, under conditions that permit said targeting oligonucleotide, including said DNA portion having at least on ribonucleotide, to hybridize to said mRNA of interest to form a hybrid between the mRNA of interest and the targeting oligonucleotide, wherein said targeting oligonucleotide is further defined as follows:
- i) said DNA portion having at least on ribonucleotide comprises or consists of four to six deoxyribonucleotides in length, wherein said DNA portion having at least on ribonucleotide is complementary to a sequence at the 5′-end portion of said mRNA of interest within 15 to 50 bases from said most 5′ end ribonucleotide;
- and
- ii) said RNA portion comprises 5 to 15 ribonucleotides in length and said RNA portion is complementary to a corresponding RNA portion in said mRNA of interest flanking the sequence hybridized to the targeting oligonucleotide's DNA portion having at least on ribonucleotide, wherein said at least one ribonucleotide is complementary to the counterpart ribonucleotide of the mRNA of interest;
- c) providing a nuclease that selectively cleaves RNA in a DNA/RNA hybrid, resulting in the cleavage of said mRNA of interest in the sequence region hybridized to the targeting oligonucleotide's DNA portion having at least on ribonucleotide and the release of:
- i) the cleaved mRNA 5′-end portion fragments with said cap structure;
- and
- ii) the cleaved mRNA 5′-end portion fragments without said cap structure;
- d) electrophoretically separating said released mRNA 5′-end portion;
- and
- e) determining the relative amount of said separated capped fragments and uncapped fragments, thereby quantifying efficiency of mRNA capping in said mRNA of interest.
The method of claim 1, wherein said mRNA of interest is produced by in vitro transcription (IVT) of a DNA template encoding said mRNA of interest, and wherein said cap structure is post- or co-transcriptionally added to the most 5′-end ribonucleotide of said mRNA of interest.
The method of claim 1, wherein said nuclease only cleaves RNA that is hybridized to DNA and said nuclease has a minimum binding domain size of four nucleotides.
The method of claim 1, wherein said nuclease cleaves said mRNA of interest only once.
The method of claim 1, wherein said electrophoretically separating comprises use of a gel that is a polyacrylamide gel selected from the group consisting of a polyacrylamide gel comprising 17% to 22% acrylamide, 20% acrylamide, and 19.5% acrylamide and 0.50% Bis-acrylamide.
The method of claim 1, wherein said released mRNA 5′-end portion fragments are fragments of 25 to 40 nucleotides in length.
The method of claim 1, wherein said targeting oligonucleotide is further defined by one of the following features or any combination(s) thereof:
- i) the Tm of said targeting oligonucleotide (7) is 40° C. to 68° C.;
- ii) the DNA portion of the targeting oligonucleotide is flanked with RNA portions on both ends;
- iii) the targeting oligo (TO) anneals to an mRNA having an autologous 5′-untranslated region (5′ UTR);
- iv) the targeting oligo (TO) anneals to a heterologous 5′-untranslated region (5′ UTR) that is used for many different mRNAs;
- v) the targeting oligo anneals to a Xenopus 5′ UTR;
- vi) said targeting oligo is designed to so the DNA portion does not hybridize to counterpart ribonucleotides in sequence at the 5′-end portion of the mRNA of interest that comprise modified ribonucleosides selected from pseudouridine and N1-methylpseudouridine;
- and
- vii) the length of the targeting oligonucleotide in terms of nucleotides is different from the released mRNA 5′ end portion fragments.
The method of claim 1, said method further comprising:
- a) providing:
- i) a collection of molecular weight markers, including a marker ladder;
- and/or
- ii) a control mixture of 5′-end capped and uncapped control mRNA fragments;
- b) parallel electrophoresing:
- said released mRNA 5′-end portion fragments;
- and said collection of molecular weight markers and/or said control mixture of 5′-end capped and uncapped control mRNA fragments;
- and
- c) comparing the length of the gel separated mRNA 5′-end portion fragments with said weight markers and/or said control mixture of 5′-end capped and uncapped control mRNA fragments.
A kit or kit-of-parts, said kit or kit-of-parts comprising:
- a) a nuclease that selectively cleaves RNA in a DNA/RNA hybrid, and
- b) a Stop/Loading buffer containing at least one dye or two dyes.
Dependencies are inferred from the claim text.