Proc. Natl. Acad. Sci. USA
`Vol. 87, pp. 1874-1878, March 1990
`Biochemistry
`
`Isothermal, in vitro amplification of nucleic acids by a multienzyme
`reaction modeled after retroviral replication
`(reverse transcriptase/RNase H/T7 RNA polymerase)
`JOHN C. GUATELLI*t, KRISTINA M. WHITFIELDt, DEBORAH Y. KWOHt, KEVIN J. BARRINGERt,
`DOUGLAS D. RICHMAN*t, AND THOMAS R. GINGERASO§
`*Departments of Medicine and Pathology, University of California San Diego School of Medicine, and tSan Diego Veterans Administration Medical Center,
`San Diego, CA 92161; and tThe Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA 92037
`Communicated by Ronald M. Evans, December 18, 1989 (receivedfor review October 6, 1989)
`
`ABSTRACT
`A target nucleic acid sequence can be repli-
`cated (amplified) exponentially in vitro under isothermal con-
`ditions by using three enzymatic activities essential to retroviral
`replication: reverse transcriptase, RNase H, and a DNA-
`dependent RNA polymerase. By mimickisg the retroviral
`strategy of RNA replication by means of cDNA intermediates,
`this reaction accumulates cDNA and RNA copies of the original
`target. Product accumulation is exponential with respect to
`time, indicating that newly synthesized cDNAs and RNAs
`function as templates for a continuous series of transcription
`and reverse transcription reactions. Ten million-fold amplifi-
`cation occurs after a 1- to 2-hr incubation, with an initial rate
`of amplification of 10-fold every 2.5 min. This self-sustained
`sequence replication system is useful for the detection and
`nucleotide sequence analysis of rare RNAs and DNAs. The
`analogy to aspects of retroviral replication is discussed.
`
`The transfer of genetic information from RNA to DNA and
`then back to RNA is a scheme characteristic of retroviruses.
`Such a scheme provides a mechanism for the replication of
`RNA genomes (reviewed in refs. 1-3). In exploring variations
`of an in vitro transcription-based amplification system (TAS)
`(4), it was discovered that it was possible to devise a
`concerted, three-enzyme, in vitro reaction to carry out an
`isothermal replication of target nucleic acid sequences, anal-
`ogous to the strategy used in retroviral replication. This
`reaction is a self-sustained sequence replication (3SR) system
`involving the collective activities of avian myeloblastosis
`virus (AMV) reverse transcriptase, Escherichia coli RNase
`H, and T7 RNA polymerase. The accumulation ofboth target
`nucleic acid-specific RNA and cDNA has been observed,
`quantitated, and characterized. Several aspects of this 3SR
`amplification protocol provide features not observed in the
`use ofeither the polymerase chain reaction or TAS protocols.
`These features and the parallels between the strategies em-
`ployed in the 3SR reaction and during retroviral replication
`are noted and discussed.
`
`MATERIALS AND METHODS
`Materials. AMV reverse transcriptase was purchased from
`Life Sciences (Saint Petersburg, FL); T7 RNA polymerase
`was from Stratagene; E. coli RNase H and RNase-free DNase
`I were from Bethesda Research Laboratories. Oligonucleo-
`tides were synthesized by phosphoramidate chemistry by
`using an Applied Biosystems model 380A DNA synthesizer.
`Trisacryl beads containing capture oligonucleotides, which
`were used for sandwich hybridizations, were prepared sim-
`ilarly to those described previously (5).
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`RNA Purification. Human immunodeficiency virus type 1
`(HIV-1) RNA was extracted in total RNA from HIV-
`1-infected CEM cells by the guanidinium isothiocyanate/
`cesium chloride gradient procedure (6) and quantitated by
`comparative hybridization to standards of known concentra-
`tion.
`3SR Reaction. One hundred-microliter 3SR amplification
`reactions contained the target RNA, 40 mM Tris-HCI at pH
`8.1, 20 mM MgCl2, 25 mM NaCl, 2 mM spermidine hydro-
`chloride, 5 mM dithiothreitol, bovine serum albumin (80
`,ug/ml), 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP,
`4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 250 ng of
`oligonucleotide 88-211 (primer B, 5'-AATTTAATACGACT-
`CACTATAGGGATCTATTGTGCCCCGGCTGGTTTTGC-
`GATTCTA-3'), and 250 ng of oligonucleotide 88-347 (primer
`A, 5'-AATTTAATACGACTCACTATAGGGATGTACTAT-
`TATGGTTTTAGCATTGTCTAGTGA-3'). [Each priming
`oligonucleotide contains the T7 RNA polymerase binding
`sequence (italic) and the preferred transcriptional initiation
`site (boldface). The remaining sequence is complementary to
`the target HIV-1 sequence.] After heating at 650C for 1 min
`and cooling at 370C for 2 min, 30 units of AMV reverse
`transcriptase, 100 units of T7 RNA polymerase, and 4 units
`ofE. coli RNase H were added to each reaction. All reactions
`were incubated at 370C for 1 hr or the amount of time
`indicated and stopped by placing the reaction on ice.
`Bead-Based Sandwich Hybridization. The 3SR amplifica-
`tion product and 32P-labeled oligonucleotide 87-81 (5'-
`AATTAGGCCAGTAGTATCAACTCAACT-3') were dena-
`tured in 30 ttl of 10 mM Tris, pH 8.1/1 mM EDTA at 650C for
`5 min in an Eppendorf tube. To this, 30 ul of a solution
`hybridization mixture [10x standard saline phosphate/EDTA
`(6)10%o (wt/vol) dextran sulfate/0.2% SDS] was added. The
`solution was mixed and incubated at 420C for 2 hr.
`Approximately 25 mg (wet weight) of Trisacryl beads
`containing oligonucleotide 86-273 (5'-AGTCTAGCAGAA-
`GAAGAGGTAGTAATTAGA-3') was prehybridized in 250
`pA of hybridization solution (5 x standard saline phosphate/
`EDTA/10% dextran sulfate/0.1% SDS) in a 2-ml microcol-
`umn (Isolab) for 30 min at 370C. The prehybridization solu-
`tion was removed, and 40 p.1 of fresh hybridization solution
`was added to the beads, together with the 60 p.1 of solution
`from the solution hybridization step. The beads were then
`incubated at 370C for 1 hr with occasional mixing and then
`washed six times with 1 ml of 2x standard saline citrate (6)
`at 370C. The radioactivity of the beads and the combined
`washes was measured by Cerenkov counting for 1 min. The
`amount of target detected was determined by calculating the
`percentage of total radioactivity captured on the beads and
`
`Abbreviations: 3SR, self-sustained sequence replication; TAS, tran-
`scription-based amplification system; HIV-1, human immunodefi-
`ciency virus type 1; AMV, avian myeloblastosis virus.
`§To whom reprint requests should be addressed.
`
`1874
`
`Ariosa Exhibit 1022, pg. 1
`IPR2013-00276
`
`

`

`Biochemistry: Guatefli et al.
`then multiplying this percentage by the femtomoles of 32P-
`labeled oligonucleotides used for the hybridization.
`Nuclease Digestion. Samples of 3SR reactions were di-
`gested with RNase-free DNase I in 50 mM sodium acetate at
`pH 6.5, 10 mM MgCl2, and 2 mM CaC12 in the presence of
`enzyme (25 pug/ml) at 370C for 1 hr. Digestions were termi-
`nated by the addition of EDTA to 12.5 mM and were
`extracted once with an equal volume of phenol/chloroform
`prior to separation in a 5% denaturing polyacrylamide gel.
`Direct 3SR Transcript Sequencing. One hundred-microliter
`3SR reactions were extracted once with an equal volume of
`phenol/chloroform, adjusted to 0.3 M sodium acetate, and
`precipitated with the addition of 2.5 volumes of 100o etha-
`nol. The reaction products were pelleted at 16,000 x g for 10
`min, rinsed with 70%o (vol/vol) ethanol, and dried completely
`before being dissolved in 50 A.l of sterile H20. Two percent
`of the transcript (derived from a 107-fold amplification from
`100 amol of target HIV) was sequenced (7) by using a primer
`complementary to the major antisense 3SR RNA product and
`located within the targeted env region (5'-GACGTTCAATG-
`GAAC-3').
`cDNA Cloning and Sequencing. The 3SR reactions were
`extracted once with phenol/chloroform, precipitated with
`ethanol, and converted into first- and second-strand cDNAs
`by the single-tube method first described by D'Alessio and
`Gerard (8). The second-strand synthesis reaction was termi-
`nated by heating, and the cDNA ends were repaired by the
`direct addition of 20 units of the large fragment ofE. coli DNA
`polymerase I, followed by a 1.5-hr incubation at 22°C.
`End-repaired cDNAs were extracted once with one volume
`of phenol/chloroform and- then precipitated (in the presence
`of 10 ,g of glycogen as a carrier) with 3 volumes of 100%o
`ethanol. Precipitated cDNAs were phosphorylated with T4
`polynucleotide kinase (6), ligated into pUC19 that had been
`cut with Sma I and treated with phosphatase, and used to
`transform E. coli strain MC1061. 3SR clone-containing trans-
`formants were identified by colony hybridization by using a
`kinased oligonucleotide probe. Clones were sequenced with
`a Sequenase dideoxy sequencing kit according to the manu-
`facturer's directions (United States Biochemical).
`
`RESULTS
`3SR Strategy and Kinetics ofAmplification. The strategy for
`RNA replication by the 3SR reaction is depicted in Fig. 1. In
`summary, a continuous series of reverse transcription and
`transcription reactions replicates an RNA target sequence by
`means of cDNA intermediates. The crucial elements of this
`design are (i) as with the TAS protocol, the oligonucleotide
`primers both specify the target and contain 5' extensions
`encoding the T7 RNA polymerase binding site, so that the
`resultant cDNAs are competent transcription templates (4);
`(ii) cDNA synthesis can proceed to completion of both
`strands due to the degradation of RNA in the intermediate
`RNADNA hybrid by RNase H; and (iii) the reaction prod-
`ucts (cDNA and RNA) can function as templates for subse-
`quent steps, enabling exponential replication. During the
`initial stage, the 3SR reaction proceeds first to the formation
`of antisense RNA, as drawn in Fig. 1. This pathway is
`attributable to the lack of a double-stranded DNA structure
`for the 17 promoter, which would produce a sense RNA
`transcript (Fig. 1, step 6). This situation predicts the produc-
`tion of antisense RNA as the favored product in the initial
`cycle of a 3SR reaction in which two 17 promoters are used.
`Such a 3SR strategy has been applied to the amplification
`of a 214-nucleotide region of the envelope gene (env) of
`HIV-1, measured by using a bead-based sandwich hybrid-
`ization system, which detects and quantitates the single-
`stranded RNA copies specific for this region of env (9).
`Amounts of HIV-1 RNA from 10-5 to 10-1 fmol were
`
`Proc. Natl. Acad. Sci. USA 87 (1990)
`
`1875
`
`5'
`
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`
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`Strategy of the 3SR scheme. The 3SR reaction depends
`FIG. 1.
`on a continuous cycle of reverse transcription and transcription
`reactions to replicate an RNA target by means of cDNA intermedi-
`ates. Oligonucleotides A and B prime DNA synthesis and encode the
`promoter sequence for the T7 RNA polymerase (black boxes). Steps
`1-6 depict the synthesis of a double-stranded cDNA, which is a
`transcription template for 17 RNA polymerase. Complete cDNA
`synthesis is dependent on the digestion of the RNA in the interme-
`diate RNA*DNA hybrid (step 4) by RNase H. Transcription-
`competent cDNAs yield antisense RNA copies of the original target
`(step 7, right). These transcripts are converted to cDNAs containing
`double-stranded promoters on both ends in an inverted repeat
`orientation (steps 7-12). These cDNAs can yield either sense or
`antisense RNAs, which can reenter the cycle. Thin lines, RNA; thick
`lines, DNA; RT, reverse transcription.
`replicated in a 2-hr 3SR reaction, and the 3SR RNA products
`were measured by bead-based sandwich hybridization sys-
`tem as a function of time (Fig. 2A). Within the first 15 min,
`each of the input HIV-1 RNA target concentrations was
`increased 105-fold, followed by a 90-fold increase over the
`next 105 min. Interestingly, during the first 10 min (Fig. 2A
`Inset), the amount of 3SR RNA products increased -10-fold
`every 2.5 min. The slower rate of replication observed after
`the first 10 min does not appear to be attributable to the
`accumulation of target-specific products of the reaction,
`since the rate of amplification decreased at the same time for
`all initial target concentrations. The initial exponential rate of
`increase in the copy number of this env region is consistent
`with a repeated cycling mechanism for the 3SR reaction in
`which the products function as templates, as predicted in Fig.
`1. In addition, the reported (10) maximal initiation rate of the
`T7 RNA polymerase (0.8 initiation per second per template)
`appears to be too slow to produce the levels of RNA product
`observed within the first 15 min of the 3SR reaction in the
`absence of an increase in the number of templates during the
`reaction. The efficiency of the 3SR reaction is affected only
`slightly by the initial concentration of HIV-1 RNA, as is
`observed when 3 x 106- and 9 x 106-fold amplifications were
`produced from 10-1 and 10-5 fmol of input target HIV-1
`RNA, respectively.
`Characterization of 3SR Amplification Products. This self-
`sustained cyclical reaction predicts the accumulation ofRNA
`products, principally due to the efficiency ofthe transcription
`steps, but also due to single- and double-stranded DNA
`copies of the original target RNA sequence. The cDNA
`products of the 3SR reaction have been characterized (Fig.
`
`Ariosa Exhibit 1022, pg. 2
`IPR2013-00276
`
`

`

`Biochemistry: Guatefli et al.
`
`Proc. Natl. Acad. Sci. USA 87 (1990)
`
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`Accumulation and quantitation of RNA and cDNA products of 3SR reactions over time. (A) The kinetics of antisense RNA
`FIG. 2.
`accumulation is demonstrated as measured by a bead-based sandwich hybridization system. Each point represents the average of four
`experiments, and the standard deviation is illustrated by the error bars. The input target, sense RNA, was quantitated by comparative
`hybridization with HIV-1 sequences, which was quantitated spectrophotometrically. (Inset) Results obtained from two 3SR reactions; the
`starting HIV-1 target RNA concentration was 1 fmol for time points of 1-5 min and 0.1 fmol for 5-15 min. The fold amplification presented at
`each time point is the average of 2-4 experiments. (B) The accumulation with time of labeled cDNA products containing either one promoter
`(245 base pairs) or two promoters (266 base pairs) in a 3SR reaction is shown. The reaction was' initiated with 50 amol of HIV-1 RNA as the
`target, with 250 ng of 32P-end-labeled oligonucleotide 88-211 (primer B) and 250 ng ofunlabeled oligonucleotide 88-347 (primer A) as the primers.
`The products were electrophoresed on a 6%6 denaturing polyacrylamide gel. The 245- and 266-base-pair bands from the 31-min reaction were
`excised, the amount of radioactivity incorporated was assayed, and the amount of labeled cDNA was calculated to be,-"5 pmol. nt, Nucleotides.
`quences (Fig. 3A). Numerous RNA products were also
`2B). cDNAs of a prescribed length, as defined by the posi-
`observed that were' less than full length but hybridized with
`tions oftwo primers,' accumulate throughout the course ofthe
`the HIV-1 env-specific probe. The fidelity of synthesis of
`reaction. When two primers containing T7 promoters were
`used in amplifying 50 amol of HIV-1 RNA and one of the 3SR
`these product RNAs to the original target sequence was
`partially verified by a direct sequencing reaction (7) using a
`prmers (primer B) was end-labeled with 32p, the cDNA
`products observed were 245 (214-base HIV sequence +
`32P-labeled internal primer (Fig. 3B), which yielded the
`27-base T7 sequence + 4-base transcription start sequence)
`expected sequence 'from the HIV-1 env region (9). Interest-
`and 266 (same as 245-base product + 21-base T7 sequence)
`ingly, when a 32P-labeled probe specific for the detection of
`bases in length (Fig. 2B). The 245-base cDNA presumably
`results from the extension of primer B on an antisense
`transcript [Fig. 1, step 9 (antisense)]. After degradation ofthe
`RNA in the RNADNA hybrid and the subsequent annealing
`ofprimer A, the cDNA can extend to the full 266-base length
`(Fig. 1, step 12).
`An estimate of the average transcriptional efficiency of the
`3SR reaction can be made by comparing the amplification of
`RNA to that of DNA. When the cDNA products containing
`one or two T7 promoters were excised from the polyacryl-
`amide gel, the amount of radioactivity incorporated was
`assayed (Fig. 2B), and the amount of amplification was
`calculated by using the specific activity of the 32P-labeled
`primers, at least 105-fold cDNA amplification could be mea-
`sured as a result of these reactions. When compared to the 9
`x 106-fold levels of RNA produced from the same type of
`reaction, this productivity in the yield of cDNA indicates
`that, on the average, each cDNA template directs the syn-
`thesis of a minimum of 90 copies of detectable RNA during
`the 3SR reaction.
`The RNA products from the 3SR reactions have been
`analyzed. In the case of the amplification of the 214-
`nucleotide region of the HIV-1 env gene, DNase digestion of
`the 3SR reaction products revealed antisense RNA that is 245
`nucleotides' in length (Fig. 3A). Full-length RNA products
`from these 3SR reactions contain the sequences complemen-
`tary to the Ti promoter sequence at the 3' end of the
`transcript (Fig. 1), so that the 245 nucleotides represent the
`combined lengths of the HIV-1 (214 nucleotides), T7 (27
`nucleotides), and transcription start (4 nucleotides) se-
`
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`Characterization of 3SR RNA and DNA products by
`FIG. 3.
`Southern hybridization and nucleotide sequencing. (A) An autora-
`diograph of a 5% denaturing polyacrylamide gel shows the results of
`a 3SR reaction after 20, 30, 45, and 60 min into the reaction, before
`and after digestion with DNase I. Digested and undigested products
`were analyzed by Southern blot analysis (6) using oligonucleotide
`88-298 as the labeled probe (5'-ACAGTACAATGTACACATG-
`GAATTAGGCCA-3'). Sizes of the bands (in nucleotides)- are indi-
`cated. (B) Direct sequencing (7) of the antisense RNA transcript from
`a 3SR reaction. The portion shown reads 5'-TGGAATTAGGCCAG-
`TAGTATCAACTCAACTGCTGTTAAA-3' and is the exact match
`for the HIV-1 sequence described by Ratner et al.'(9)'from residues
`6548 to 6586.
`
`Ariosa Exhibit 1022, pg. 3
`IPR2013-00276
`
`

`

`Proc. Natl. Acad. Sci. USA 87 (1990)
`
`1877
`
`in part, from RNase H degradation during the 3SR reaction.
`When products from a 60-min 3SR reaction were reincubated
`with a-labeled CTP and 17 RNA polymerase and chased with
`unlabeled CTP, then RNA that was 245 nucleotides in length
`(full length, including a T7 promoter complementary se-
`quence at the 3' end) was produced (Fig. 4B). If the products
`were reincubated with T7 polymerase and E. coli RNase H,
`a truncated RNA of -200 nucleotides was produced. The
`truncated RNA presumably results from degradation of the 3'
`end of the molecule; when annealed to the DNA oligonucle-
`otide primer, a substrate for RNase H (RNA-DNA hybrid) is
`formed. Curiously, the entire length of this RNA-DNA du-
`plex was not degraded; if it were, the RNA molecule would
`be rendered unusable as a template for subsequent cDNA
`synthesis.
`The concept that many 3SR RNA products have end
`truncations that delete primer-complementary sequences is
`supported by a comparison of Figs. 3A and 2B. RNA prod-
`ucts detected by hybridization with an internal probe (Fig.
`3A) revealed several species of short products, which may
`represent both incomplete transcripts as well as degradation
`products.
`
`DISCUSSION
`As a practical amplification strategy, the 3SR protocol is
`suited to the specific detection and quantification of RNA
`B
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`Biochemistry: Guatelli et al.
`sense-strand RNA was used in a hybridization, 100-fold less
`sense-strand RNA was detected than antisense RNA (data
`not shown). The mechanism by which one promoter assumes
`the greatest production of RNA is partially explained by
`measuring the transcriptional efficiency of each promoter
`independently. The results of this measurement indicated an
`8-fold difference in the ability of the two promoters to
`produce RNA transcripts from this region, in favor of the
`antisense T7 promoter (data not shown).
`Nucleotide sequence characterization of the products of a
`3SR reaction reveals truncated products. Ten individual
`clones derived from the RNA and cDNA ofthis reaction have
`been analyzed, and they contain both complete and truncated
`forms of the predicted sequence (Fig. 4A). Three of these
`clones (97, 116, and 125) appear capable of perpetuating the
`cycle of 3SR replication. Although it is not possible to
`determine the degree to which these truncations resulted
`from cloning artifacts versus the amplification process, such
`shorter products are seen in Fig. 3. Two variations in the
`sequence of the 3SR clones (121 and 128) are noted when
`compared to the original HIV-1 sequence ofthis region. Such
`variations from the parent sequence are presumably the
`result of the mismatch error properties of both the reverse
`transcriptase and T7 polymerase.
`Influence ofE. coli RNase H on RNA Products of3SR. RNA
`products that are less than full length appear to result, at least
`A
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`FIG. 4. The effect ofE. coliRNase H during a 3SR reaction and nucleotide sequence analysis of cloned 3SR products. (A) A map of 10 isolated
`and sequenced cDNA clones derived from a 60-min 3SR reaction is illustrated. The targeted portion of the HIV-1 env region is shown at the
`top of the figure (9). The env region is demarked by primers 88-211 and 88-347, each of which contains 27 base pairs (bp) of a consensus T7
`RNA polymerase binding sequence (PBS), and the corresponding target-complementary sequences (TCS). Differences between the cloned
`sequences and the published sequences are indicated. (B) Products of a 60-min 3SR reaction (0.1 pmol), which amplified 107-fold (from 0.1 amol),
`were reincubated in 3SR buffer containing 0.5 mM GTP, 0.5 mM ATP, 0.5 mM UTP, 6 jAM unlabeled CTP, 10 ,uCi of [a-32P]CTP (800 Ci/mmol;
`1 Ci = 37 GBq) as well as 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dTTP, and 0.5 mM dCTP. The reactions were heated to 65°C for 1 min and
`cooled to 37°C for 1 min prior to the addition of 100 units of T7 RNA polymerase and 4 units of E. coli RNase H (lane A), 30 units of AMV
`reverse transcriptase and 100 units of T7 RNA polymerase (lane B), or 100 units of T7 RNA polymerase (lane C). The reactions were incubated
`for 15 min at 37°C, followed by the addition of 0.1 nmol of unlabeled CTP, with continued incubation at 37°C for another 15 min. After removal
`of the unincorporated label, 1%o of the reaction was resolved on a 6% denaturing polyacrylamide gel before autoradiography. Sizes of the bands
`(in base pairs) are indicated.
`
`Ariosa Exhibit 1022, pg. 4
`IPR2013-00276
`
`

`

`1878
`
`Biochemistry: Guatelli et al.
`sequences. The in vitro 3SR reaction operates at 370C and,
`consequently, temperatures required to denature double-
`stranded DNA are not employed, unlike both the TAS (4) and
`polymerase chain reaction (11) protocols. Under these con-
`ditions, double-stranded DNAs would fail to function as
`amplification templates, rendering 3SR ideal for assessing the
`transcriptional activity of specific genes. However, DNA
`templates have been amplified by using this 3SR reaction,
`with efficiencies equivalent to those observed for RNA
`templates (data not shown) by the inclusion of a denaturation
`step at the outset of the protocol. A second feature ofthe 3SR
`reaction distinct from the well-used polymerase chain reac-
`tion protocol is the rapid kinetics observed in the 3SR
`reaction. To achieve the 105-fold amplification observed in
`the first 15 min of the 3SR reaction, a polymerase chain
`reaction would require 85 min, even if one assumes a per-
`cycle time of 5 min and a per-cycle efficiency of 100%.
`Also of practical importance is the finding that 3SR can
`operate effectively if only one primer contains the T7 pro-
`moter (data not shown). The effectiveness of 3SR reactions
`employing only one primer encoding the T7 promoter indi-
`cates that, in contrast to the genomic RNA of retroviruses, it
`is not necessary for 3SR RNA products to encode a promoter
`sequence on their 3' ends. This 3' RNA structure is required
`for retroviral replication in order to solve the problem of
`promoter-sequence loss during transcription (12). In the 3SR
`reaction, this RNA structure is unnecessary because the
`primers themselves bring the promoter sequence into the
`cDNA molecule.
`The in vitro exponential amplification of a region of the
`HIV-1 genome using three enzymatic activities that are found
`in all cells infected with retroviruses suggests that the accu-
`mulation of multiple viral DNA copies found in such cells
`may be due to an intracellular, 3SR-like mechanism. In
`chronically infected cells and biopsied lymph node tissue,
`HIV-1 accumulates long-lived, unintegrated, viral DNA cop-
`ies, predominantly in linear forms (13). The presence of such
`multiple copies of unintegrated viral DNA has also been
`reported in duck cells infected in vitro with B77 (14), spleen
`necrosis virus-infected avian cells (15), avian leukosis virus-
`infected cells (16, 17), as well as in bone marrow cells of cats
`infected with a feline leukemia virus variant that causes an
`AIDS-like illness (18). The accumulation of such uninte-
`grated viral DNA copies has been correlated with the cyto-
`pathic effects observed for avian leukosis virus (16); in feline
`AIDS, the appearance of a variant form of unintegrated DNA
`correlates with the onset of disease (18). The origin of
`high-copy-number, unintegrated viral DNA is unknown, but
`viral DNA can be derived from either multiple infections of
`the same host cell or the reverse transcription of newly
`transcribed, full-length viral RNA. The multiple reinfection
`of the same host cells is an unlikely explanation for the
`accumulation of HIV-1 viral DNA in vivo because of the low
`number of cells infected and their low level of RNA expres-
`sion (19). Intracellular cycles of reverse transcription and
`transcription, however, could result in viral DNA accumu-
`lation, even with low-level RNA expression, by means of the
`process exemplified by the in vitro 3SR reaction.
`The parallel between the in vitro 3SR reaction and the
`production of viral DNA also opens to speculation the role of
`RNase H in both of these systems. Retroviral RNase H is
`considered to be the enzyme responsible for the digestion of
`RNADNA hybrids, so that second-strand cDNA synthesis
`can occur. For murine leukemia virus, replication is depen-
`dent on reverse transcriptase-associated RNase H (20). Yet,
`neither AMV nor murine leukemia virus RNase H can
`efficiently catalyze the 3SR reaction without supplementa-
`tion by E. coli RNase H (data not shown). Traditionally,
`retroviral RNase H has been believed to be a processive
`exonuclease, requiring free ends (21-23), although recent
`
`Proc. Natl. Acad. Sci. USA 87 (1990)
`
`1.
`
`2.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`evidence suggests endoribonuclease activity as well (24). E.
`coli RNase H is an endoribonuclease (25). By analogy to the
`E. coli RNase H dependency of 3SR, it is interesting to
`speculate on the role that the host-cell RNase H, an endo-
`ribonuclease like that of E. coli (22), may play in retroviral
`replication.
`The early evolution of reverse transcriptase activity has
`been proposed as an important event during the transition from
`an early-RNA to present-day DNA world (26). The coupling of
`reverse transcription to transcription creates a system for
`information transfer between two classes of macromolecules,
`RNA and DNA. The ease with which the repetitive cycling of
`this process can occur in vitro, catalyzed by only three
`enzymes, suggests that it may have played an early role in the
`evolution of nucleic acid replication strategies.
`We thank A. W. McCue and N. Riggs for their technical assist-
`ance, L. Blonski and C. Lynch for the synthesis and purification of
`the oligonucleotides used, and J. Doty for her careful preparation of
`this manuscript. This work was supported in part by Public Health
`Service Grants HB-67019, A152578, and NIH 5-T32-AI07036 from
`the National Institutes of Health and by the Veterans Administration
`(D.D.R.) and the Salk Institute Biotechnology/Industrial Associ-
`ates, Inc. (T.R.G.).
`Varmus, H. E. & Swanstrom, R. (1984) inRNA Tumor Viruses, eds.
`Weiss, R., Teich, N., Varmus, H. & Coffin, J. (Cold Spring Harbor
`Laboratory, Cold Spring Harbor, NY), Vol. 1, pp. 369-512.
`Varmus, H. E. & Swanstrom, R. (1984) inRNA Tumor Viruses, eds.
`Weiss, R., Teich, N., Varmus, H. & Coffin, J. (Cold Spring Harbor
`Laboratory, Cold Spring Harbor, NY), Vol. 2, pp. 75-185.
`Varmus, H. E. (1988) Science 240, 1427-1435.
`3.
`4. Kwoh, D. Y., Davis, G. R., Whitfield, K. M., Chappelle, H.,
`DiMichele, L. & Gingeras, T. R. (1989) Proc. Natl. Acad. Sci. USA
`86, 1173-1177.
`Ghosh, S. S. & Musso, G. F. (1987) Nucleic Acids Res. 15, 5353-
`5372.
`Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular
`Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,
`Cold Spring Harbor, NY).
`Stoflet, E. S., Koeberl, D. D., Sarkar, G. & Sommer, S. S. (1988)
`Science 239, 491-494.
`D'Alessio, J. M. & Gerard, G. F. (1988) Nucleic Acids Res. 16,
`1999-2014.
`Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B.,
`Josephs, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, E. A.,
`Baumeister, K., Ivanoff, L., Petteway, S. R., Pearson, M. L.,
`Lautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T.,
`Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 313, 277-
`284.
`Martin, C. T. & Coleman, J. E. (1987) Biochemistry 26, 2690-2696.
`Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T.,
`Erlich, H. A. & Arnheim, N. (1985) Science 230, 1350-1354.
`Gilboa, E., Mitra, S. W., Goff, S. & Baltimore, D. (1979) Cell 18,
`93-100.
`Shaw, G. M., Hahn, B. H., Arya, S. K., Groopman, J. E., Gallo,
`R. C. & Wong-Staal, F. (1984) Science 226, 1165-1171.
`Varmus, H. E. & Shank, P. R. (1976) J. Virol. 18, 567-573.
`Keshet, E. & Temin, H. M. (1979) J. Virol. 31, 376-388.
`Weller, S. K. & Temin, H. M. (1981) J. Virol. 39, 713-721.
`Weller, S. K., Joy, A. E. & Temin, H. M. (1980) J. Virol. 33,
`494-506.
`Mullins, J. I., Chen, C. S. & Hoover, E. A. (1986) Nature (London)
`319, 333-336.
`Harper, M. E., Marselle, L. M., Gallo, R. C. & Wong-Staal, F.
`(1986) Proc. Natl. Acad. Sci. USA 83, 772-776.
`Repaske, R., Hartley, J. W., Kavlick, M. F., O'Neill, R. R. &
`Austin, J. B. (1989) J. Virol. 63, 1460-1464.
`Grandgenett, D. P. & Green, M. (1974) J. Biol. Chem. 249, 5148-
`5152.
`Keller, W. & Crouch, R. (1972) Proc. Natl. Acad. Sci. USA 69,
`3360-3364.
`Verma, I. M. (1975) J. Virol. 15, 843-854.
`Krug, M. S. & Berger, S. L. (1989) Proc. Natl. Acad. Sci. USA 86,
`3539-3543.
`Leis, J. P., Berkower, I. & Harwitz, J. (1973) Proc. Natl. Acad. Sci.
`USA 70, 466-470.
`Varmus, H. E. (1989) Cell 56, 721-724.
`
`14.
`15.
`16.
`17.
`
`10.
`11.
`
`12.
`
`13.
`
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`24.
`
`25.
`
`26.
`
`Ariosa Exhibit 1022, pg. 5
`IPR2013-00276
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