CROSS REFERENCE TO RELATED APPLICATION

This claims priority to European Patent Application No. EP 18205148.2, filed Nov. 8, 2018, which is hereby incorporated by reference herein in its entirety for all purposes.

FIELD

The present invention relates to a method of characterizing a target RNA polynucleotide.

BACKGROUND

The general goal of transcriptomics is to fully sequence all transcriptional species in a sample, determine the transcriptional structure of a gene, including splice variants and fusion genes, quantify changes in the expression levels of transcripts at different stages of development and in different environments, and determine antisense transcription occurs in biological cells. The most widely used strategies for next-generation RNA-sequencing include poly-T priming or ribonucleic acid (RNA) fragmentation followed by random hexamer initiation of synthetically complementary DNA (cDNA). These cDNA strands are then usually prepared for analysis on a given sequencing system using library preparation methods that include PCR. However, which type of PCR is used in the preparation of a library will introduce bias. For example, the resulting library will be less complex than the total amount of mRNA because not all transcripts will be amplified with the same efficiency, leading to the exit of certain RNA species and over-amplification of other species. In addition, PCR amplification generates a synthetic copy of the original RNA strand and potential epigenetic information may be lost. Therefore, there is a clear need for largely avoiding the amplification of RNA molecules during library preparations for sequencing purposes.

Exceptions to PCR-based library preparations include FRT-Seq on the Illumina platforms, where the first-strand cDNA synthesis reaction is performed on single strands of RNA hybridized to the flow cell surface. Another example is direct RNA sequencing on the Helicos platform, which is a sequencing-by-synthesis reaction using native RNA strands as a template. In both cases, PCR amplification is avoided, removing this source of distortion, thus the data obtained more faithfully reflect the abundance of RNAs in the original sample. However, these approaches are still based on synthetic copies of the original RNA strand so that information about modifications is not preserved. Both approaches, in addition, necessarily produce short sequence reads, which is somewhat disadvantageous since transcripts in eukaryotes typically undergo alternative splicing, producing several different isoforms, e.g., with different transcription start sites, coding sequences, and untranslated regions. Short read sequencing, however, is typically unable to span entire transcripts and may miss new splice variants.

A new direct RNA sequencing approach has been developed by Oxford Nanopore Technologies. This approach, which is also depicted inFIG. 1is based on an annealing of a poly-T primer to a poly-A-tail of mRNA molecules. It hence only and exclusively works with RNA molecules having a poly-A tail and excludes all other RNA molecules.

There is hence a need for an improved direct RNA sequencing methodology allowing to sequence all RNA species, i.e., also those RNA molecules which do not possess a poly-A-tail.

SUMMARY

The present invention addresses this need and provides a method of characterizing a target RNA polynucleotide comprising: (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv); (vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and (vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore, wherein the measurements are indicative of one or more characteristics of the RNA polynucleotide, thereby characterizing the target RNA polynucleotide.

The present invention also provides a kit for characterizing a target RNA polynucleotide, as well as a use of a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form for the modification of an RNA polynucleotide.

The presented method, being based on random multimer sequences, advantageously allows to perform direct RNA sequencing with all RNA species, not only those which comprise poly-A-tails, i.e., mRNAs. Accordingly, also mRNA fragments of different origins may be sequenced. The stem-loop polynucleotide further allows for a stabilization of the transcript and allows for the presentation of the random multimer at the 3′ end of the stem-loop polynucleotide. Further, the presence of a restriction enzyme recognition site in the stem-loop polynucleotide further allows for a cleavage of the polynucleotide yielding a 3′ A overhang, which can subsequently be used for the connection of an adaptor polynucleotide complex associated with an RNA translocase enzyme necessary for translocating the RNA molecule through a transmembrane pore in order to obtain its sequence.

In a further aspect, the present invention relates to a method of preparing a target RNA polynucleotide for transmembrane sequencing comprising: (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; and (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv).

In one embodiment of the above mentioned methods, the polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form is a DNA polynucleotide comprising one or more non-DNA nucleotides.

It is preferred that the polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form comprises one or more 5-methyl-isoC-isoG base-pairs.

In a further embodiment, the 5-methyl-isoC-isoG base-pairs are provided at least at every 4th, 5thor 6thposition of the stem-loop polynucleotide.

In yet another embodiment, the 5-methyl-isoC-isoG base-pairs are provided at least at every 4th, 5thor 6thposition of the stem-loop polynucleotide.

In a further embodiment of the present invention, the random multimer segment is a random hexamer, random heptamer, random octamer, random nonamer or random decamer segment. It is preferred that the random multimer segment is a random hexamer segment.

According to another embodiment of the present invention, the polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form comprises a restriction enzyme recognition site of the sequence 5′-ACAGT-3′ or 5′-TCAGA-3′.

In a further embodiment, the enzyme Hpy188I is used to cleave the restriction enzyme recognition site 5′-TCAGA-3′; or the enzyme Bst4CI, HpyCH4III or TaaI is used to cleave the restriction enzyme recognition site 5′-ACAGT-3′.

In yet another embodiment, the polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form additionally comprises a barcoding section.

In another embodiment of the present invention, the one or more characteristics of the RNA polynucleotide which are measured during the translocation of the RNA polynucleotide through the transmembrane pore, are (i) length of the RNA polynucleotide, (ii) identity of the RNA polynucleotide, (iii) sequence of the RNA polynucleotide, and (iv) presence of a nucleotide modification in the RNA polynucleotide. It is preferred that the characteristic to be measured is the sequence of the RNA polynucleotide.

In a further embodiment, the RNA polynucleotide is any RNA polynucleotide. In specific embodiments, it is mRNA, a fragment of mRNA, pre-mRNA, ncRNA, rRNA, miRNA, snoRNA, srRNA, tmRNA, siRNA or piRNA.

In another embodiment according to the present invention, the RNA translocase is SF2 helicase, NS3 helicase, NPH-II helicase, Upfl helicase or RIG-I translocase or derivatives thereof, or a derivative of a DNA helicase such as Hel308 helicase, RecD helicase, XPD helicase or Dda helicase which is capable of translocating RNA polynucleotides.

According to a further embodiment, the transmembrane pore protein is a pore protein derived from hemolysin, leukocidin, MspA, MspB, MspC, MspD, CsgG, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA.

In another aspect, the present invention relates to a kit for characterizing a target RNA polynucleotide comprising a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form, wherein the stem-loop form comprises one or more 5-methyl-isoC-isoG base-pairs, preferably comprising a polynucleotide with a 3′ terminal random multimer segment and having a stem-loop form as defined herein.

In yet another aspect, the present invention relates to a use of a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form, wherein the stem-loop form comprises one or more 5-methyl-isoC-isoG base-pairs for the modification of an RNA polynucleotide, thereby preparing the RNA polynucleotide for a direct RNA sequencing through a transmembrane pore.

It is to be understood that the features mentioned above and those yet to be explained below may be used not only in the respective combinations indicated, but also in other combinations or in isolation without departing from the scope of the invention.

