There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.
Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.
When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current change of known signature and duration. In the strand sequencing method, a single polynucleotide strand is passed through the pore and the identities of the nucleotides are derived. Strand sequencing can involve the use of a polynucleotide binding protein to control the movement of the polynucleotide through the pore.
Messenger RNA provides a view of the dynamic state of an organism and the benefits and applications of direct RNA sequencing are vast, including use in health screening; for example metastasis progression in certain cancers and heart disease. Direct RNA sequencing also has applications in investigating disease resistance in crops, determining the response to stresses, for example; drought. UV and salinity and in cellular differentiation and determination during emblyogenesis.
A problem which occurs in direct sequencing of RNA, particularly those of 500 nucleotides or more, is finding a suitable molecular motor which can control translocation of the RNA through a transmembrane pore. To date, a molecular motor that engages with RNA and provides consistent movement has not been shown. Consistent movement of the RNA polymer and the ability to read long segments of the polymer is desirable for characterising or sequencing the polynucleotide.
International Patent Application No. PCT/GB2014/053121 (WO 2015/056028) describes a method of characterising a target ribonucleic acid (RNA) involving forming a complementary polynucleotide and then characterising the complementary polynucleotide using a transmembrane pore. Such indirect RNA characterisation is prone to error and can result in the loss of vital information regarding, for example, methylation status of the RNA. Other important modifications can also be hidden in the conversion of RNA to cDNA.