The 1960s, 1970s and 1980s were both the early years and golden years for nucleic acid enzymology. Scientists unraveling the secrets of DNA replication and repair, RNA transcription, viral replication and other basic processes purified enzymes responsible for numerous processes. Other scientists envisioned practical applications for these enzymes and put them to work in the recombinant DNA revolution that began just over 50 years ago. Due in no small part to the great body of literature that has arisen around these pioneer enzymes, they tend to be important still today in biotechnology - sometimes retaining monopolies on a particular type of in vitro biochemistry. But there are new entrants, and today I’m going to explore a new player in the reverse transcription space, UltraMarathonRT from a small Connecticut company, RNAConnect. RNAConnect has launched two new products in the second half of this year, a kit for cDNA synthesis back in August and today a kit for generating long direct RNA reads on Oxford Nanopore platforms.
Two reverse transcriptases have dominated the field of cDNA generation for cloning, sequencing, RT-qPCR and other applications, both arising from early research on retroviruses. Each is named for the retrovirus it was found in, Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV). Many of the reverse transcription kits on the market today are either formulations of one of these two retroviruses or made with versions carrying a small number of point mutations. While these enzymes have served biotechnology well, they do have shortcomings, particularly in the lack of helicase activity which can cause them to stall on templates which have formed complex and strong secondary structures.
UltraMarathonRT is not from a eukaryotic retrovirus, but instead from a prokaryotic group II self-splicing intron. UltraMarathonRT is far more processive than the venerable eukaryotic RTs and able to reverse transcribe transcripts of 30 kilobases or longer. A key difference from another group II retron RT on the market, Induro from NEB, is that UltraMarathonRT has a temperature optimum of 30C vs 55C for Induro; higher temperatures risk more damage to template RNA.
Template switching is a property of reverse transcriptases in which the enzyme has a 3’ terminal transferase activity which adds a predictable set of untemplated nucleotides to the 3’ end of the first strand cDNA; for UltraMarathonRT this is three As. By inclusion in the reaction of an oligo with the complementary sequence, second strand cDNA can be triggered in the same reaction. Since the template switching oligo (TSO) can have barcode and unique molecular identifier sequences as well, this makes template switching particularly valuable for sequencing applications.
RNAConnect today launched a kit for Oxford Nanopore direct RNA sequencing which uses UltraMarathonRT. While direct RNA sequencing can work without any reverse transcription, having a first strand cDNA bound to the RNA improves performance, particularly since the helicase activity of UltraMarathonRT can unwind secondary structures which might not be unwound by the motor protein in ONT’s chemistry. RNAConnect has run comparisons of their kit to a process using Induro and shown 67% more mapped reads which are in excess of 10Kb, with this dropping to 24% for mapped reads >5kb and only 11% for reads >2kb. With increasing research interest in exploring long non-coding RNAs (lncRNAs) - the best known lncRNA, XIST responsible for driving X chromosome inactivation, is 17 kilobases long. The kit also has the convenience of including all required components, other than those unique to ONT.
Higher processivity and greater ability to push through complex secondary structures - both highly desirable properties in a reverse transcriptase. As the price/performance ratio of both PacBio and ONT improve for cDNA sequencing, continued improvement of metrics for ONT direct RNA and now meso-length reads from Roche’s SBX chemistry will all enable greater surveys of RNA at longer scales. Such studies can be reasonably expected to sharpen our understanding of splicing.
When I was an undergraduate nearly 40 years ago, we were taught that alternative splicing existed but was a rare phenomenon that only rarely deserved attention. I think we were taught about the alternative splicing that drives soluble vs membrane-bound antibodies in B cells, but probably no other examples. That view has changed radically over the last 40 years, with alternative splicing now recognized as a generator of both protein diversity and regulation.
As a graduate student, I discovered an overlooked set of alternative exons in a Drosophila visual protein gene which my labmate Carlos Alvarez demonstrated, by clever PCR assays, that while there are theoretically 8 different splice forms possible (which would all generate valid ORFs) only three of these are detectable in flies. Made a nice PNAS paper. Nowadays we’d do that by sequencing. Cataloging such coordinated splicing would be one clear use for long read direct RNA or long read cDNA sequencing.
Another emerging class of splicing events of great interest are “poison exons” and “detained introns”. Detained introns are introns that are normally spliced out, but are systematically retained in certain contexts. If these cause the translation of a premature stop codon which leads to nonsense-mediated decay of that mRNA, then it is a poison exon. A number of labs have reported on poison exons that appear to be very carefully regulated, providing yet another opportunity for cells to regulate production of a particular gene product. Inadvertent retention of poison exons is yet another way that mutations can negatively affect mRNAs and trigger rare genetic disorders.
Clearly for a complete survey of alternative splicing, poison exon usage, and other types of retained/detained introns it is important to have an unbiased view of a transcript, not degraded by secondary structure or biased to the 3’ end. UltraMarathonRT shows approximately 2X higher detection of retained introns than other RTs when applied to the Universal Human Reference RNA (UHRR) sample.
RNAConnect the company is sited in Branford CT, not far from Yale University where founder Dr. Anna Marie Pyle teaches. For this piece I spoke with Andrew Bond, previously at gene synthesis company Gen9, and Jason Underwood, ex-PacBio,
UltraMarathonRT appears to be a useful new tool in the molecular biology toolbox, enhancing the ability to sequence long and difficult RNA templates. It shows promise for advancing the understanding of splicing as well as the medical consequences of inappropriate splicing. UltraMarathonRT citations in PubMed are currently only from Dr. Pyle’s lab; it will be interesting to see what new discoveries are made as kits with this enzyme enter widespread use in RNA sequencing laboratories.
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