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WO2023196738A1 - Séquençage de nanopores d'arn à l'aide d'une transcription inverse - Google Patents

Séquençage de nanopores d'arn à l'aide d'une transcription inverse Download PDF

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Publication number
WO2023196738A1
WO2023196738A1 PCT/US2023/064680 US2023064680W WO2023196738A1 WO 2023196738 A1 WO2023196738 A1 WO 2023196738A1 US 2023064680 W US2023064680 W US 2023064680W WO 2023196738 A1 WO2023196738 A1 WO 2023196738A1
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rna
nanopore
sequence
sequencing
bmrt
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Susan MARQUSEE
Alan Shaw
Kathleen Collins
Carlos J. Bustamante
Heather UPTON
Sydney PIMENTEL
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Definitions

  • the structure of RNA is vital to its biological function3, and therefore methods to assign structure have been developed using the standard Illumina sequencing platform4,5.
  • these methods indirectly sequence cDNA generated from reverse transcription and can so only aim to infer RNA structure from altered fidelity or termination of the sequenced DNA.
  • the invention provides systems, methods and compositions for direct nanopore sequencing of RNA using a cellular reverse transcriptase to capture and thread single-stranded RNA through an MspA nanopore without requirement of ligation or prior conversion to cDNA.
  • the invention can determine RNA secondary structures simultaneously with direct sequencing. RNA sequence and structure can be determined at the single-molecule level in scalable nanopore sequencing, enabling the determination of the relation between RNA function and structure for any RNA even in a complex RNA pool.
  • the invention can be utilized to directly probe and sequence RNA from biological, clinical, or research samples without the need of prior amplification, ligation, or conversion to cDNA, therefore reducing potential biases that these extra steps introduce to the original sample.
  • the invention can be used to detect RNA secondary structures in the samples during sequencing.
  • the invention is used to directly capture and sequence full-length RNA extracted from biological samples, such as tissue or cell culture, or to assay the sequence homogeneity of supposedly identical RNAs generated in vitro or in vivo from a single type of DNA template.
  • the invention outputs the sequence and secondary structure information of the RNA sample.
  • Some embodiments of the invention deploy primers that are tagged with cholesterol to be hybridized to target RNA.
  • Other variations utilize the “template jumping” activity of the particular cellular reverse transcriptase employed, whereby the enzyme can directly bind to the 3’ end of RNA regardless of sequence and initiate reverse transcription.
  • the reverse transcriptase will be immobilized on the lipid membrane via a lipid anchor, and free RNA inside the sample well can be captured and thread through the nanopore for direct RNA sequencing.
  • the invention may also be practiced in alternative embodiments, including alternative RTs, such as cellular selfish element RTs, alternative RNA inputs, which may be entirely or partly non-single-stranded, and alternative nanopores.
  • alternative RTs such as cellular selfish element RTs
  • alternative RNA inputs which may be entirely or partly non-single-stranded
  • nanopores alternative nanopore sequencing using a biological nanopore, MspA, which has a smaller length of RNA in the constricted region of the pore and therefore fewer and more resolved current signatures necessary to define to relate current to sequence.
  • the invention uses a physiological motor with high processivity tracking on RNA template and operably low impedance by RNA structure or modified nucleotides or chemical damage.
  • the invention uses a membrane-tethered DNA primer to recruit RNA by base-pairing, which in one example can be visualized as depicted in Fig.1A.
  • RNA 3’ can enter pore but RT-bound duplex cannot.
  • RNA is copied into cDNA by the RT, the template that the RT has copied over is pulled away through the pore. (RT back side is butted up to the pore; the pore gets fed the already-copied RNA.)
  • RNA is sequenced 3’ to 5’.
  • the invention uses a membrane-tethered RT to recruit RNA template base-paired to DNA.
  • RNA 3’ can enter pore but duplex cannot.
  • RNA is copied into cDNA by the RT, the template that the RT has copied over is pulled away through the pore.
  • RNA is sequenced 3’ to 5’. This can be visualized with reference to Fig. 1A, by changing the tether to be on the RT rather than the DNA primer.
  • the invention uses the RT in terminal transferase buffer to 3’ extend molecules in the input RNA pool with a nucleotide or nucleotide analog or combination thereof, either in one step or in two separate steps to add two sequential tail sequences.
  • RNA is sequenced 3’ to 5’.
  • the invention uses membrane-tethered DNA primer to base pair with the RNA 3’ end. RNA 5’ can enter pore but duplex cannot. Add RT to initiate synthesis. As RT uses template for cDNA synthesis, RNA is pulled out of the pore. (RT front side is butted up to the pore). RNA is sequenced 3’ to 5’. This can be visualized with reference to Fig.
  • RNA template + annealed portion of primer relative to the pore (RT on same side of pore).
  • the invention uses membrane-tethered RT bound to primer to catch an RNA 3’ end near the pore, not necessarily by base-pairing.
  • the RNA 5’ end can pass through the pore until it is halted by resistance from RT grip on the 3’ end. Initiate synthesis to pull the template RNA out of the pore.
  • RNA is sequenced 3’ to 5’. This can be visualized with reference to Fig.
  • the invention is applied to sequencing of DNA or other nucleic acid like molecules or chimeric nucleic acid like molecules; [019] the invention RT motors additionally engineered for desired performance; [020] the invention is used with no need for prior knowledge of RNA or DNA 3’ end sequence; [021] the invention is used with no need for RNA ligation (although ligation could be used to add handle(s) to input RNA); [022] the RNA structure does not induce motor dissociation or impose a lasting barrier; [023] the MspA nanopore quadromer map enables high accuracy of sequencing; [024] the invention provides information about RNA structure simultaneously with sequencing; [025] the invention can develop RNA quadromer map that includes discrimination of modified nucleotides; and/or [026] the invention is configured for automation and high-throughput.
  • the invention provides an engineered cellular reverse transcriptase as a potent motor protein that can processively thread ssRNA through the MspA biological nanopore in single nucleotide steps while it is synthesizing cDNA. Threading ssRNA through the MspA nanopore in discrete steps, and ssRNA sequencing with the MspA nanopore, are novel aspects of the invention. Using ssRNAs of known sequences, and we constructed the “quadromer map” for ssRNA in the MspA nanopore, a table that can convert measured nanopore ion current to RNA sequences. In addition, we demonstrate that the single-molecule kinetic rates of the reverse transcriptase are affected by the presence of stable RNA secondary structures.
  • the invention enables commercial RNA sequencers that can directly sequence RNA extracted from any biological sample.
  • the technology can be used to identify expression levels, mutations, secondary structures, and chemical modifications of RNA.
  • the long read nature of nanopore sequencers enables sequencing full length RNA without the need of fragmentation.
  • the invention provides an RNA sequencing system as shown in Fig.1, and/or as described herein.
  • the invention provides a method of nanopore sequencing of RNA using reverse transcription comprising: using an engineered cellular reverse transcriptase as a motor to directly capture and processively thread single-stranded RNA through a MspA porin nanopore in single nucleotide steps while it is synthesizing cDNA, without requirement of ligation or prior conversion to cDNA.
  • the reverse transcriptase is a truncated, modified Bombyx mori R2 non-LTR retroelement RT (BoMoC without or with amino acid substitution(s)), e.g.
  • the nanopore porin comprises MspA of Mycobacterium smegmatis.
  • the invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.
  • Fig. 1A-B Sequencing RNA with a eukaryotic RT and MspA nanopore.
  • A The instrument setup of the MspA nanopore sequencer.
  • a lipid bilayer was generated to separate two wells containing buffer solutions, a single MspA nanopore was inserted into the lipid membrane, and a bias of 140 mV was applied to the system during data acquisition.
  • Template RNA blue line
  • DNA primer red line
  • RT After introduction of the RT, it forms an elongation complex that can get captured by the nanopore.
  • the RT will come into rest on top of the nanopore and by cDNA synthesis will continuously thread RNA into the pore in discrete steps.
  • the positions of the polyA sequences are not to scale due to illustration purposes.
  • Top and middle panel Ion current signal from the translocation of RNA1 and RNA1_polyA.
  • RNA1_polyA has two polyA sequences inserted close to the 5’ of the RNA about 80 nt apart (top panel, highlighted in orange) while RNA1 does not have polyA inserted (middle panel).
  • the orange rectangles highlight the regions where polyA is inserted, and as seen in the top panel, polyA insertion gave rise to high ion current signal (top panel) that does not exist in the data without polyA insertion (middle panel, orange rectangles).
  • Bottom panel The blue line represents a segment of the normalized ion current levels from the middle panel, along with RNA sequence aligned to the ion current signals. [037] Fig.
  • RNA secondary structures by analyzing the single molecule kinetics of bmRT.
  • A. Top panel Overlay of a segment of raw nanopore ion current from RNA translocation (blue) and the steps found via a point of change algorithm8 (grey). Single steps can be detected, and their individual dwell times can be quantified by fitting the cumulative distribution function (CDF) of the dwell time of the same step obtained from difference RNA translocation traces to a single exponential (bottom panel).
  • CDF cumulative distribution function
  • Bottom panel Dwell time distribution of the first sequence A repeat and second sequence A repeat overlayed, the sequence underneath represent the sequence in the enzyme’s catalytic site at every step. Sequence A is highlighted in magenta and the remainder of the terminal hairpin is in black.
  • C. Top panel: a 32 nt RNA oligonucleotide (short black line) was hybridized to the RNA template.
  • Bottom panel Dwell time distribution comparison between the same RNA sequence with (red line) and without (blue line) hybridization of the RNA oligonucleotide. Error bars are 95% confidence interval. [038]
  • Fig. 3A-C An active helicase model to describe the helicase activity of bmRT. A.
  • translocation by one step would require that the –3 nucleotide becomes unpaired.
  • the helicase can sense RNA structures both at the –3 position and at further downstream nucleotides up to position –13 or –14 (total length of 11-12 nt), possibly due to preferential binding of the helicase to ssRNA.
  • C Model prediction for the dependence of overall translocation rate as a function of the average base pair stability in downstream RNA. The sigmoidal becomes sharper with increased length (m) of the downstream sensing region following position -3.
  • Fig. 4A-B Detecting Broccoli RNA-BI ligand binding using direct RNA nanopore sequencing.
  • A sequence design of RNA3_Broccoli, the G bases involved in GQ formation are highlighted in red.
  • a 6nt RNA duplex that precedes GQs is highlighted in blue. Information about which G bases are involved in GQ formation is obtained from ref. 25.
  • B Average dwell time of bmRT along the Broccoli RNA sequence.
  • the Broccoli RNA sequence is highlighted in orange and its upper and lower GQ is marked in magenta and grey respectively.
  • the BI binding site is located on top of the upper GQ.
  • a strong pause was observed when the bmRT’s catalytic site is at nt # 32, which is 12 nt from the start of the lower GQ and 2 nt from the start of the dsRNA duplex.
  • Fig. 5 The MspA nanopore instrument setup. Two wells (cis and trans wells) are separated by an insulating lipid bilayer. A single MspA nanopore protein (yellow object cross section) is inserted into the nanopore in the “backwards” fashion.
  • FIG. 6A-B The longest Ion current traces obtained by using Eubacterium rectale RT (A left panel) and Bombyx mori RT (B left panel) and RNA1 shown with different time scales (X axis).
  • a and B right panel Alignment of the ion current traces to the consensus ion current sequence for the RNA1 (construction of the consensus ion current is described in Supplementary Note 1).
  • RNA1 The consensus ion current sequence of RNA1is shown in blue, and ion current levels from A and B are shown in orange. Alignment of the ion current levels to the consensus ion current sequence is achieved by methods developed by the Gundlach lab8. The length of the individual read in (A left panel) and (B left panel) is shown in orange traces and numbers. [042] Fig. 7. Examples of the end of RNA translocation events. Events consistently end after the final tall peak for this particular template, and the current measured upon no further RNA translocation is consistently about 45 pA. RNA1 was used to generate this data. [043] Fig. 8A-E. Construction of RNA ion current consensus sequence. A. Ion current vs. RNA sequence.
  • RNA2 was used to generate this data.
  • Fig. 10A-D Stopping of RNA translocation in the MspA nanopore as bmRT reaches the 5’ end of the template.
  • A in the raw traces acquired (an example from RNA1 is shown), we observed that bmRT fluctuates between two states when it reaches the end of the template, the origin of this fluctuation is unclear.
  • B nucleotide position where bmRT stops (we used the first of the two states bmRT fluctuates between at the stop position) at the end of RNA1.
  • bmRT was observed to stop at positions ranging from nt 359 to nt 364 in the nanopore, with stopping at nt 360 being the major product.
  • bmRT extends its cDNA product by synthesis of a variable length of 3’ overhang21 , and this non-templated addition (NTA) of zero to five nt could allow additional ssRNA entry to the nanopore.
  • NTA non-templated addition
  • Fig. 11 Dwell time distribution of the RT along an RNA template with two barriers. The enzyme exhibits long dwell times when it encounters stable dsRNA.
  • GGGUG For the broccoli RNA, after the initial canonical duplex (CGCCUC), an energy of –3kcal/mol is used for each of the next five nucleotides (GGGUG).
  • This segment corresponds to nucleotides stacked on the duplex, part of the bottom mixed tetrad, and the first G of the lower G quadruplex29. (A good fit in panel D is not sensitive to the exact energy value used for this segment which can be between –2.5 and –3.5 kcal/mol per nucleotide, and the length of this segment can be four to six nucleotides.
  • the grey box highlights sites that have high dsRNA % but fast translocation rate.
  • the dots are color coded to show GC content.
  • C. mFold predicted structures of RNA3 in region 1 and 2. In region 1, sites 67 to 69 have high dsRNA % ahead but have fast translocation rate. From the structure we can see that these sites are in an internal loop of a hairpin, and the hairpin should be unfolded when bmRT arrives at these sites, and the opened hairpin no longer poses as a barrier to translocation.
  • bmRT translocation rate after the initial invasion the hairpin no longer slows down the enzyme.
  • Fig. 15A-C bmRT kinetics on bare RNA3.
  • A. The average dwell time (red) of bmRT on a segment of RNA3, containing the 5’ terminal hairpin. The minimum rate is indicated by straight blue line.
  • B. Total base pairing energy (black curve, left axis) and individual base pairing energy (dashed red curve, right axis) as in Figure 13A-D, assuming full complementarity.
  • Calculated per-nucleotide total base pairing probability in the thermodynamic ensemble (extracted from probability matrix depicted in Fig. 18) is shown as dotted lines for interactions with the 5’ side (P5’, magenta) or 3’ side (P3’, blue).
  • the solid red line is the probability-scaled base pairing free energy, defined as ⁇ G bp ⁇ (w1+w2.P5’ +w3.P3’) where w1 to w3 are empirical weights and the term inside the parentheses is capped at one.
  • C Model prediction for the translocation profile using the probability-scaled base pairing free energy given in panel B, with the parameter values indicated.
  • Fig. 16 Binding of Broccoli RNA aptamer to ligand BI. Top panel: image of ligand titration in test tube. Fluorescence intensity increases until saturation as the ratio of ligand increases from 0 to 10-fold. Bottom panel: quantification of fluorescence intensity by Image J. Fluorescence intensity reaches saturation when ligand is in 5-fold excess. Measurement was repeated three times and plotted with error [052] Fig. 17. Predicted bmRT helicase power as a function of average base pair stability in the downstream region (of length m). The output power curves for three values of m are shown.
  • Fig. 18 Structure dot plot showing possible base pairs in RNA3 predicted by mFold. See Supplementary Note 3.
  • the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
  • RNAs are hybridized to a short DNA primer that is tagged with cholesterol to direct the RNA/DNA complex to the nanopore membrane ( Figure 1A). Addition of the RT results in binding of the enzyme to the RNA/DNA junction and consequent initiation of cDNA synthesis.
  • RNA 3’ end of an elongation complex is drawn through a backwards inserted MspA nanopore ( Figure 1A , Figure 5) and the RT comes to rest on top of the pore, preventing additional RNA transit through it.
  • the rate of cDNA synthesis by RT dictates the 3’-to-5’ passage of the RNA template chain through the pore in discrete steps until synthesis is complete.
  • the MspA nanopore technique is also a powerful tool to dissect the biophysical function of molecular motors8 as the exact position of the motor protein on its template can be determined, and its single-molecule biophysical parameters (such as dwell time, pauses, backtracking activity) at every single step on the template can be detected and analyzed.
  • Establishment of MspA nanopore sequencing of RNA is also a powerful tool to dissect the biophysical function of molecular motors8 as the exact position of the motor protein on its template can be determined, and its single-molecule biophysical parameters (such as dwell time, pauses, backtracking activity) at every single step on the template can be detected and analyzed.
  • Nanopore sequencing requires two main components: 1) a processive motor to either pull or feed single-stranded (ss) DNA or RNA through the nanopore in discrete steps, and 2) an a priori knowledge of the ion currents corresponding to all the possible sequences that partially block the nanopore.
  • ss single-stranded
  • MspA nanopore which has been previously exploited for DNA9 and peptide10 sequencing.
  • a major challenge to our goal was to identify a motor protein that could translocate the RNA template through the MspA nanopore in a processive and controlled manner.
  • RNA sequences used in this study are summarized in Supplementary Table 1.
  • Construction and Validation of the MspA RNA Quadromer Map A nanopore sequencing approach requires a library of currents corresponding to all the possible sequences that can be found inside the nanopore. For the MspA nanopore, this correlation is referred to as the “quadromer map”8 since the ion current is determined by the 4 nt that span the constriction site of the pore. Because RNA sequencing with the MspA nanopore had not yet been achieved, we first proceeded to obtain the RNA quadromer map for the MspA nanopore.
  • RNA1 (Supplementary Table 1)
  • RNA sequencing traces consistently ended with a very similar signature followed by RNA signal stillness in the pore ( Figure 7), which coincides with bulk biochemistry observations that bmRT does not readily dissociate from its RNA template upon completion of cDNA synthesis14. Therefore, we could assume that the ion current signals obtained close to the end of a translocation event originate from sequences close to the 5’ end of the RNA.
  • each nucleotide in the RNA template can be assigned to a single step in the consensus ion current series confirms that the bmRT takes single-nucleotide steps on its RNA template and sequentially releases a single nt of RNA at a time to enter the nanopore.
  • the nanopore reports on the RNA sequence partially blocking the current through the constriction site of the pore. In order to relate the dwell times of the RT with the presence of RNA structures in front of the enzyme (next sections), we need to know the exact location of the enzyme on the RNA template when a particular sequence is in the pore.
  • RNA structure via nanopore sequencing kinetics Detection of RNA structure via nanopore sequencing kinetics.
  • Stable RNA secondary structures have been shown to affect the kinetic rates of molecular motors such as the ribosome16, RNA helicases17, and retroviral RTs18,19.
  • To extract kinetic information corresponding to the RNA sequence we determined the average dwell time before each RT step on the RNA template. This procedure involved pooling data obtained for that step from multiple sequencing traces of the same sequence and fitting them to a single exponential function (Figure 2A).
  • RT kinetic profiles obtained in the presence and absence of the hybridized oligonucleotide showed pauses in translocation at distinct positions within the dsRNA region ( Figure 2C bottom panel, and Figure 11).
  • dwell times with and without the dsRNA barrier remained similar for most translocation steps.
  • bmRT dwell times at most positions do not appear to be changed by the presence of the secondary structures in the RNA either in the form of a hairpin or in the form of a duplex. Rather, the enzyme seems to pause at certain particular positions in this region and be unaffected in the regions of secondary structure that surround them. This behavior suggests that bmRT functions as an active helicase capable of destabilizing RNA structures 20 . To explain why the enzyme slows down at certain specific locations within the secondary structures, we constructed an active helicase model to quantitatively describe the kinetic profile of bmRT as a function of barrier stability.
  • the translocation cycle of bmRT consists of a residence phase during which events such as dNTP binding and catalysis occur, followed by a stepping phase in which the motor attempts to move along its track.
  • the overall observed dwell time at each position would equal k resid -1 + k step -1 , where k resid and k step are the rates of completing a residence and the rate of stepping of the enzyme, respectively.
  • the observed dwell times in the presence of barriers can be well explained if the stepping rate depends not only on the base pairing stability of the nucleotide that is stepped over (at the helicase site of the enzyme), but also on the stability of several downstream nucleotides: k step ⁇ P u P mu k ss (1) where P u is the probability that the stepped-over nucleotide is in its unpaired state, P mu is the probability that the following downstream segment of length m is in its unpaired state, and k ss is the stepping rate over single-stranded RNA (in the absence of barrier).
  • P u is a function of the Gibbs free energy difference between the unpaired and paired states of the nucleotide: where ⁇ is (k B T) -1 , ⁇ Gb P is the free energy of base pairing for the nucleotide, and ⁇ Ga is the destabilization energy due to the helicase. A large negative value of ⁇ Ga would represent a more “active” helicase 20 .
  • m is the length of the downstream segment following the stepped-over nucleotide, is the total base-pairing free energy of the downstream segment, and ⁇ Ga is the same as above (per nucleotide).
  • the value of ⁇ Gb P at each nucleotide position can be estimated precisely using the nearest neighbor rules 22 (as the difference in the ⁇ G of the barrier before and after opening of the given nucleotide). Additionally, k resid can be determined from the observed translocation rates in the absence of the barrier. This leaves k ss , ⁇ Ga, and m as the only free parameters in the model. After fitting these parameters, dwell times predicted using Eqs. 1 to 3 are in excellent agreement with the kinetic profiles obtained in the presence of different barriers, with the major and minor points of slowdown properly reproduced (Figure 3A and Figure 13D).
  • Downstream sensing could be mediated by direct interaction of the helicase with the RNA to destabilize its folding 17,19,23 , or by a mechanism in which the kinetic stability of the junction arises from a long-range allosteric coupling through the double helix 24 .
  • RNA is known to spontaneously form secondary structures of short polymer lengths 25
  • longer dwell times are only observed in front of downstream RNA regions that have high probability of being double-stranded, most of which have high GC content (Figure 14A-C).
  • Figure 15A-C the predicted base pairing probabilities into our active helicase model, we can qualitatively reproduce the observed pattern of dwell times with an overall correlation coefficient of ⁇ 0.6
  • RNA3_Broccoli in Supplementary Table 1 RNA template that contains a single Broccoli RNA aptamer 27 at its 5’ end. This aptamer has two G-quadruplexes (GQ) and can bind the fluorescent ligand BI, which stabilizes the folding of the aptamer RNA 27,28 .
  • the Broccoli GQs are preceded by a short RNA duplex (Figure 4A) which was shown to be important in folding of the aptamer based on sequence truncation experiments 27 .
  • Figure 4A We first showed that the aptamer binds to BI under our experimental conditions ( Figure 16). Using our assay, we then compared the single-molecule kinetic profiles of bmRT on Broccoli RNA with and without the presence of BI ( Figure 4B). Results indicate that binding to BI and stabilization of the Broccoli RNA structure led to a significant pause of the bmRT when the helicase site of the enzyme is still 1 nt away from the start of the Broccoli RNA duplex.
  • RNA template preparation RNA template sequences were ordered as dsDNA gBlocks that contain the T7 promoter from Integrated DNA Technologies (IDT) and inserted into a linearized pRZ plasmid using the infusion cloning kit (Thermo Fisher) and transformed into Sure2 cells (Agilent) following manufacturer’s instructions. Positive colonies were screened with Sanger sequencing.
  • RNA oligonucleotide and DNA primer with 5’ cholesterol modification were ordered from IDT.
  • RNA template was mixed with DNA primer (and when relevant a 10-fold excess of RNA oligonucleotide for dsRNA barrier experiments) to a final concentration of 0.8 ⁇ M and 2 ⁇ M respectively, in buffer containing 20 mM Tris pH 8.0 and 20 mM NaCl and heated to 75°C for 90 seconds and immediately placed on ice until further use.
  • DNA primer and when relevant a 10-fold excess of RNA oligonucleotide for dsRNA barrier experiments
  • the open reading frame of the enzymes was codon optimized and ordered from GenScript, and inserted with an N-terminal maltose binding protein tag into the MacroLab vector 2bct that contains a C-terminal 6xHis tag (https://qb3.berkeley.edu/facility/qb3-macrolab/).
  • the enzymes were expressed in Rosetta2(DE3)pLysS cells in 2xYT medium and induced with isopropylthio- ⁇ -galactoside.
  • the MspA nanopore instrument is a custom-built instrument based on the design from the Gundlach lab8.
  • 2 wells of about 120 ⁇ l in volume were drilled into a Teflon block and the two wells were connected with Teflon tubing.
  • One end of the tube was heat-shrunk and a small hole (about 20 um in diameter) was created using a fine surgical needle.
  • Electrodes were prepared by inserting an Ag/AgCl pellet in heat shrink tubing.
  • the Teflon block was mounted onto a custom-made aluminum block. Under the aluminum block is a Peltier that is connected to a temperature control unit (TED200C, Thorlabs).
  • An Axopatch 200b (Molecular Devices) was connected to the electrodes and used to apply voltage and measure ion current.
  • the Axopatch 200b is connected to a PC using National Instrument’s data acquisition card (DAQ) and controlled with a custom LabVIEW code.
  • DAQ National Instrument’s data acquisition card
  • the well that contains the 20 um hole is referred to as the cis well, and is where all the biochemical components are introduced during sequencing data acquisition.
  • the other well is referred to at the trans well.
  • Nanopore Experiments The two wells and tubing were first filled with standard experiment buffer (40 mM HEPES pH 7.5, 400 mM KCl). 180 mV was applied to the system.
  • Broccoli RNA template sequences were ordered as dsDNA gBlock as above and inserted into a linearized pRZ plasmid using infusion cloning kit (Takara Bio) and transformed into Stellar cells following manufacturer’s instructions. Positive colonies were screened with Sanger sequencing. PCR was used to amplify templates for in vitro transcription with T7 RNA polymerase (NEB). The RNA product was extracted with phenol and concentration was measured by Nanodrop spectrophotometer (Thermo Fisher). Ligand for Broccoli RNA aptamer BI (LuceRNA) was prepared in 50mM DMSO and further diluted in water.
  • Binding of the ligand to the RNA template was tested by varying the ratio of ligand to RNA in buffer containing 20 mM Tris pH 8.0 and 20 mM NaCl and heated to 75°C for 90 seconds and immediately placed on ice. The fluorescence intensity was quantified using ImageJ. In the nanopore experiment using BI ligand, 1:15 ratio of RNA to ligand was used. [080] Data Processing. The data processing pipeline is based on methods described previously8. In short: raw data (collected at 50 kHz) was down sampled to 2 kHz, and RNA translocation events were identified by using a custom GUI written in MATLAB. A point of change algorithm8 was used to identify steps within a continuous series of RNA translocation events.
  • RNA ion current consensus sequence Our goal was to construct the consensus of ion current states observed with the RT for the RNA sequence listed in Supplementary table 1. Because RNA threaded through MspA pore in the 3 ⁇ backwards pore orientation has not been observed previously, we devised an experiment in which ‘bookends’ of poly-adenine sequence flanked a sequence of interest.
  • the AGbp for each base pair can be estimated using the nearest neighbor parameters 22 (Figure 13B).
  • the calculated dwell times in the presence of the barrier ( Figure 13C) simply mirror the base-pair stabilities, and no combination of parameters k ss and AG d can qualitatively reproduce the measured rates.
  • the stepping rate would depend not only on the probability that the immediate nucleotide is unpaired, but also on the probability that the downstream segment is unpaired; in the simplest form, we can write: k step — P u P mu k ss , where k ss and P u are the same as above, and P mu is the probability that the downstream segment is in the unpaired state: where m is the length of the downstream segment following the immediate nucleotide, is the sum of the base-pairing free energies of the m nucleotides in this downstream segment, and ⁇ G d is the same as above (per nucleotide).
  • the output power of the bmRT helicase can be approximated as the unwinding work divided by unwinding time, i.e., - ⁇ G bp k step .
  • the output power as a function of average ⁇ G bp as predicted by this model is shown in Figure 17. It can be seen that that for barriers with moderate AGb P , higher values of m result in higher output power.
  • ⁇ G dNTP the free energy released upon dNTP incorporation (and PPi hydrolysis).
  • the model presented here has a minimal number of assumptions and free parameters to avoid overfitting.
  • the model may be tuned or include additional terms.
  • the ⁇ G d may be fine grained along the downstream region to better capture the physical reality of the downstream interaction.
  • RNA molecules in equilibrium are partitioned in various folded structures.
  • mFold we can obtain the probability of every base in the RNA molecule that is double stranded across all alternative predicted RNA structures.
  • mFold predicted five different structures (shown in structure dot plot in Figure 18) and the percentage of dsRNA in the next 3-13 nt downstream to the bmRT catalytic site is calculated per predicted structure. Note that as RT progresses on a single RNA template, upstream RNA will enter the pore and is no longer able to pair with downstream regions.
  • RNA molecules that are anchored to the nanopore membrane have a high local concentration and it is possible that our technology is detecting both intra- and inter-molecular RNA structures.

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Abstract

La présente invention concerne des systèmes, des procédés et des compositions pour le séquençage direct de nanopores d'ARN à l'aide d'une transcriptase inverse cellulaire pour capturer et guider directement de l'ARN simple brin à travers un nanopore MspA sans exigence de ligature ou de conversion préalable en ADNc. En outre, l'invention concerne la génération de la séquence d'ARN par référencement des courants ioniques vers une carte quadromère d'ARN nanopore qui connecte chaque courant ionique détecté avec une seule séquence à 4 nucléotides couvrant le nanopore.
PCT/US2023/064680 2022-04-05 2023-03-19 Séquençage de nanopores d'arn à l'aide d'une transcription inverse Ceased WO2023196738A1 (fr)

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