Nucleic acid hybridization assays employing dA-tailed capture probes

ANALYTICALBIOCHEMISTRY

181,3k%f%9

(1989)

Nucleic Acid Hybridization Capture Probes

Assays Employing

dA-Tailed

I. Multiple Capture Methods David V. Morrissey,’ Massimo Lombardo, E. Patrick Groody, and Mark L. Collins Gene-Trak

Received

Systems, 31 New York Avenue,

January

John

Framingham,

K. Eldredge, Massachusetts

Press, Inc.

Hybridization assays with the targets immobilized on filters require overnight incubations with labeled probes (l-3). The nucleic acids generally have to be purified by phenol extraction prior to immobilization, especially when nonradioisotopic detection is used (4-6). Accordingly, the detection of nucleic acids from impure samples has been limited for the most part to radioisotopic methaddress:

R. Kearney,

01701

19,1989

A quantitative hybridization assay termed “reversible target capture” is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe “ternary complex” is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol(lO0 fg) of target with “P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as few as six Listeria cells have been detected in the presence of 1.25 X 10’ control cells. 0 19~9 Academic

’ Present

Kevin

Wesleyan

University,

0003-2697/89 $3.00 Copyright &? 1989 by Academic Press, All rights of reproduction in any form

Middletown,

CT 06457.

ods (7,8). To circumvent these limitations, “sandwich hybridization” assays were developed (g-11) in which the target nucleic acid is “sandwiched” between a capture probe immobilized on a solid support and a labeled probe which is usually hybridized with the target in solution. The filters used in the original format have been replaced with other solid supports such as polypropylene and polystyrene (12-14) and most recently affinity methods have been introduced to improve the speed of the capture process (12-20). All of these formats employ just one capture step. The nonradioisotopic sensitivity with unpurified nucleic acids is between 0.1 and 10.0 fmol of target for these single irreversible capture schemes (( 12-20) and this paper) with 2- to 4-h formats. Two single-step sandwich hybridization formats were described elsewhere (14) in which the target nucleic acid is sandwiched on poly(dT) polystyrene between a dAtailed capture probe and a labeled probe. Polystyrene was used as the solid phase to maximize the convenience of the manual version of the assay with a total assay time of only about 2-3 h (20 samples). In addition, the use of polystyrene as a solid phase made it possible to automate the steps of the assay readily. One of the formats described also allowed simultaneous tests to be performed on a single specimen. The formats are particularly well suited to the quantitation of RNA targets in crude specimens. The nonradioactive detection limit was about 0.2 fmol of target in specimens with the rapid assay format (14). In this paper the single capture sandwich hybridization assay employing the d&dT affinity pair (14) has been modified to take advantage of the background reduction capabilities of reversibly capturing the target out of the impure samples. With the substantially lower backgrounds, reversible target capture (RTC)2 can ’ Abbreviations and key terms use& ternary complex, an entity consisting of the target nucleic acid hybridized simultaneously to the capture probe and the labeled probe; RTC, reversible target capture, a 345

Inc. reserved.

346

MORRISSEY

ET

AL,

quantitatively detect less than 1 amol of target without prior purification of the nucleic acids in the sample. MATERIALS

AND METHODS

Materials Oligonucleotides were prepared by standard phosphoramidite methods and purified by reverse-phase HPLC. The sequence of the Listeria-specific 16 S rRNA oligonucleotide capture probe No. 773 was 5’ TGT CCC CGA AGG GAA AGC TCT GTC TCC AGA GTG GT 3’. EDTA, dextran sulfate-5000, fraction V bovine serum albumin (BSA), sodium lauroyl sarcosine (Sarkosyl), ethyldimethylaminopropyl carbodiimide (EDAC), lysozyme, cacodylic acid, and mutanolysin were from Sigma. Unlabeled nucleotides, dAIZ, and dTIZ were from Pharmacia and labeled nucleotides were from New England Nuclear. A riboprobe kit was purchased from Promega and a T4 polynucleotide kinase kit from Bethesda Research Labs. Acetylated BSA was from Bethesda Research Labs. Bronopol, an antimicrobial, was from Inolex (Philadelphia, PA). 2-Mercaptoethanol was from Bio-Rad. Guanidinium thiocyanate (GuSCN) was from Fluka (98% pure or better). Polynucleotide homopolymers and terminal deoxynucleotidyl transferase were from Life Sciences, Inc. (10,000 units of terminal deoxynucleotidyl transferase/ml or higher). COOH-derivatized ferromagnetic particles were obtained from Advanced Magnetics, Inc. (Cambridge, MA). All other chemicals were obtained from the indicated suppliers and were reagent grade or better. Magnetic separators were from Corning. GeneScreen nylon membranes were from New England Nuclear. Hybrislots were from Bethesda Research Labs. Teflon acrodiscs (0.2 pm) were from Gelman. Transfer pipets were from Samco. Buffers

Used in Reversible Target Capture

The key buffers employed in this and the following study are defined in Table 1. Preparation

TABLE

Buffers

of dA-Tailed

Probes

Tailed probes were prepared essentially as described (21). A tailing mixture was prepared consisting of 0.1 M process whereby a ternary complex is purified by cycles of capture/ release from different solid supports; GuSCN, guanidinium thiocyanate; Td, dissociation temperature, under a defined set of conditions, the temperature at which one-half of the original hybrids formed (between the target nucleic acid and the capture probe) has been melted off the targeti Sarkosyl, sodium lauroyl sarcosine; NSB, nonspecific binding of labeled probe to a solid phase in the absence of target or capture probe; NSH, nonspecific hybridization, resulting from the formation of a partially mismatched hybrid between one of the probes and a sequence closely related to the targeti BSA, bovine serum albumin; EDAC, ethyldimethylaminopropyl carbodiimide.

Buffer prehybridization

Bead

wash

Bead

storage elutiona

Thermal

elution’

Filter

elution

Capture

0.1 M Tris-HCl (pH 7.5), 0.01 M EDTA, 4% fraction V BSA, 0.5% sodium lauroyl sarcosine, 0.05% bronopol 0.5 M GuSCN, 0.04 M Tris-HCl (pH 7.5), 0.008 M EDTA, 0.5% fraction V BSA, 0.5% sodium lauroyl sarcosine, 0.05% bronopol Bead prehybridization buffer plus 0.02% sodium azide 2.5 M GuSCN, 0.2 M Tris-HCl (pH 7.5), 0.04 M EDTA, 0.1% acetylated fraction V BSA, 1 pg/ml denatured E. cd DNA, 0.5% sodium lauroyl sarcosine 0.5 M NaCl, 0.1 M Tris-HCl (pH 7.5), 0.01 M EDTA, 0.5% sodium lauroyl sarcosine, 1 pg/ml denatured E. cob DNA, 0.1% acetylated BSA Same as thermal elution; alternatively, same as chemical elution if chemical elution immediately precedes filter capture

prehybridization

a These

Target Composition

Bead

Chemical

1

Used in Reversible

buffers

can be used

interchangeably.

potassium cacodylate (pH 7.0), 0.01 InM oligonucleotide, 2 mM dATP, 4 mM MgCIZ, 1 mM 2-mercaptoethanol, 0.1 mg/ml acetylated BSA (Bethesda Research Labs), and 1000 units/ml of terminal deoxynucleotidyl transferase. The reaction was performed overnight at 34OC. Preparation

of Oligo(dT)

Magnetic Beads

A. Linkage of oligo(dT) to magnetic beads functionaliced with COOH. 5’-Amine-terminated oligo(dT)rJ was synthesized by standard phosphoramidite chemistry. It was linked as described (22) to COOH-derivatized ferromagnetic particles (Advanced Magnetics, Inc.) using 0.1 M EDAC, a water-soluble carbodiimide (Sigma), to activate the COOH groups. After synthesis, the beads were washed well with sterile water and stored at 4OC in 10 mM EDTA (pH 7.0), 0.02% sodium azide, and 0.05% bronopol. B. Pretreatment of beads to reduce nonspecific binding of 1abeLzd probe, The oligo(dT) magnetic beads were pretreated twice before use. Beads were rinsed several times with water, diluted to 0.1% solids (10X dilution of the Advanced Magnetics stock concentration) with bead prehybridization buffer (Table 1) and incubated at 6WC in a Pyrex bottle with occasional vigorous agitation for 3-4 h. The beads were allowed to cool overnight with little or no agitation. The next day they were rinsed three times with sterile water and resuspended at 0.1% solids in bead prehybridization buffer plus 0.02% sodium azide at room temperature. The beads were kept rotating

d&TAILED

CAPTURE

on a tube rotator at room temperature prior to use. The beads have been stored in bead prehybridization buffer plus 0.02% azide at room temperature for up to 9 months before use with no apparent loss in function. The beads were washed once with prehybridization buffer on the day of use and resuspended in prehybridization buffer at the appropriate concentration (see next section) for target capture. Determination of the dA12 Binding Okgo Magnetic Beads

Capacity of the

Serial dilutions of the magnetic beads were made in bead prehybridization buffer and equilibrated at 37°C. The concentration range employed was from 10 to 0.3 mg/ml solids. Each aliquot of beads (0.05 ml) was mixed with 0.125 ml of 32P-labeled dA12 (5 pg/ml in magnetic bead wash buffer) or 32P-labeled dT12 (control) and incubated at 37OC for 5 min. The beads were washed twice and the dAIZ (or dT& was eluted in 2.5 M GuSCN at 37’C and counted. The beads were resuspended in wash buffer and their ODssO was determined. A plot of picomoles of dAIZ that eluted from the beads against their ODss,, was made and the binding capacity was calculated from the initial straight line portion of the curve. In a typical bead lot, one ODss,, of beads contained about 9.7 * 2.0 pmol of dTlb. One milligram of blocked solid phase equals 7.5 ODss,, (versus water); thus there were about 73 pmol of dTld per milligram of solid phase in a typical bead lot. Determination of the Stability of Poly(dA)-Oligo(dT) and Poly(rA)-Oligo(dT) as a Function of GuSCN Concentration A knowledge of the stability of poly(dA)-oligo(dT) in the capture, wash, and elution buffers is crucial to the application of this method. The stability of the poly(dA)-oligo(dT) hybrid was measured in one of two ways. In the first, the ability of poly(dA) to bind to oligo(dT) beads in various buffers was measured. P01y(dA)~~ or dA-tailed capture probes were 5’-end labeled with 32P. Poles,, (0.1 pmol) was incubated for 5 min in a total volume of 0.1 ml with oligo(dT) beads containing 1.2 pmol of oligo(dT) at 37’C in the presence of various concentrations of GuSCN. The buffers also contained 0.1 M Tris-HCI (pH 7.5), 0.01 M EDTA, 0.5% Sarkosyl, 0.05% bronopol, 0.5% fraction V BSA. After magnetic separation, the fluid was removed and discarded. The beads were incubated two times for 2 min each with these same buffers at 37’C and the fluid was discarded. The beads were resuspended in 0.6 ml bead wash buffer and scintillation counted. As a point of reference, capture was performed in 0.5 M NaCl. Poly(rA) was similarly performed with 5’-end-labeled except that 0.025 pmol of polymer was wMr.Q400, added to the beads.

PROBES,

I

347

In the second method, poly(dA) was bound to the beads in the presence of 0.5 M NaCl as above and then washed two times for 2 min each with various buffers at 37’C. Poly(rA) was treated similarly. Preparation of a Generic Eubacterial rRNA-Labeled Probe To study the detection of particular eubacterial species in crude specimens with RTC, a generic eubacterial riboprobe was prepared that could be used to hybridize to all 16 S eubacterial rRNAs. Bacteria are then sorted according to genus with genus-specific oligonucleotide capture probes. The generic riboprobe was prepared by cloning a 710nucleotide fragment from the 3’-half of the Escherichia coli 16 S rDNA into a pGEM3 vector. Riboprobes were then prepared according to the manufacturer’s (Promega) instructions. Unless indicated otherwise, the 770mer 32P-labeled probes were labeled to a specific activity of about 5-10 X 10’ cpm/pg (2.4-4.8 X lop4 cpm/molecule). Unless indicated otherwise, the labeled ribroprobe was used at 0.27 pg/ml (1.0 nM). Preparation of Bacterial Cell Extracts for Reversible Target Capture Bacterial cells were processed in one of two ways (14). In the first method for Gram-negative cells such as Campylobacter jejuni and E. coli, 9 vol of 5 M GuSCN, 0.4 M Tris-HCl (pH 7.5), 0.08 M EDTA, 1.0% Sarkosyl (Sigma) was added to 1 vol of cells (lO”/ml) and the cells were vortexed vigorously for 30 s. Cell lysis was judged to be acceptable if the optical density (after correction for dilution) at 550 nm decreased by about 95% after mixing in the GuSCN. Cell extracts were diluted to 2.5 M GuSCN prior to storage at -20°C. In the second method for Gram-positive cells such as L. monocytogenes, prior to addition of GuSCN, the following components were added to the culture medium (final concentrations): Tris-HCl (0.1 M, pH 7.5), EDTA (10 mM), lysozyme (2 mg/ml), and mutanolysin (100 units/ml). The cells were incubated for 10 min at 37°C. Sodium lauroyl sarcosine was added to 0.5%, proteinase K (Boehringer-Mannheim) was added to 1 mg/ml, and the samples were incubated for 10 min at 37OC. One volume of the 5 M GuSCN buffer containing 1.0% Sarkosyl was added. Samples were aliquoted and stored at -2W’C until use. Preparation

of Poly(dT) Nylon

Nylon filters (GeneScreen) were cut to size for the Hybrislot (BRL). Poly(dT)4Wo was filtered through a Teflon acrodisc (0.2 pm) and diluted to 0.5 mg/ml in 1.5 M NaCl, 0.5 M MgCl2,0.2 M Tris-HCl (pH 8.0). Six microliters (3 wg) was spotted onto each slot. Vacuum was applied and

348

MORRISSEY

0.2 ml of a 17:l:l:l mixture of ethanol:methanol:isopropanohwater was used to dry the filters (23). Optionally, for tighter binding of the poly(dT), the alcohol-dried filters were treated with uv (254 nm) light at 650 pW/cm* for 2 min (24). The filters were stored with desiccant at room temperature protected from light for up to 3 months before use. Preblocking of Filters and Determination of the dA12 Binding Capacity of the Poly(dT) Filters The filters were prehybridized by filtering in succession two 0.2-ml aliquots of filter prehybridization buffer (Table 1). 32P-labeled dA12 (1 pg) was filtered through in 0.2 ml of filter prehybridization buffer (0.1 ml/min). The filters were washed by filtration of 0.1 ml of filter prehybridization buffer. Blank control filters bound less than 1% of the radioactivity of the poly(dT) filters. Filters typically had a capacity of 600-1000 ng of dAIZ. Determination of the Optimal Temperature and Salt Concentration for Hybridization of Capture Probe and Target The optimal hybridization temperature and salt depend on the strength of the secondary structure in the target region and on the dissociation temperature of the “weak link” in the ternary complex, which is by design the hybrid between the oligonucleotide capture probe and the target. For 30- to 40-mer probes, the optimal hybridization temperature and salt are generally about 37°C and 2.5 M GuSCN. Values from 22-45’C should be checked at 35’C intervals with 2.5 M GuSCN. Occasionally, stronger hybridization signals are achieved with the low salt buffers. Values of 50-70°C, e.g., would be appropriate temperatures to check with 0.5 M GuSCN or 0.5 M NaCl buffers and 30- to 40-mer capture probes. Two sets of hybridizations were set up: a control set and an experimental set. Each sample contained 0.1 ml. One femtomole of target nucleic acid was used per tube. Control samples contained either no target, no tailed probe, or a heterologous tailed probe or a heterologous target. Labeled riboprobe (0.1 pmol) (or 5 pmol of labeled oligonucleotide) and 5 pmol of capture oligonucleotide were used. Hybridization was for 15 min at the various salt concentrations and temperatures. Capture was performed at 37’C for 5 min with the addition of 2 vol (0.2 ml) of magnetic beads (in bead prehybridization buffer) containing 10 pmol of bound oligo(dT). Optimally, there should be at least 0.8-1.0 M GuSCN or the equivalent concentration of NaCl in the final capture mixture. Concentrations of GuSCN higher than 1.0 M are not recommended for capture at 37°C since poly(dA-oligo(dT) hybrids begin to dissociate above that level (Appendix A). The signal/noise ratio was plotted against the various salt and temperature conditions;

ET

AL,

the optimal salt and temperature the highest signal/noise ratio.

are those which yield

Determination of Optimal Capture Probe and Labeled Probe Concentrations The optimal hybridization temperature and salt were determined first as explained above. The optimal oligonucleotide capture probe concentration was then determined at the optimal temperature and salt as follows. Again, two sets of hybridizations were set up: a control set and an experimental set. Each sample contained 0.1 ml and 1 fmol of target nucleic acid per tube. Control samples were identical with experimental except that they contained either no target, no tailed probe, a heterologous tailed probe or preferably a heterologous target. Labeled riboprobe (0.1 pmol) or 5 pmol of labeled oligonucleotide was used. The level of capture probe was generally varied between 0.1 and 5 pmol per tube. Hybridization was for 15 min in the optimal salt at the optimal temperature. Capture was performed at 37°C for 5 min with the addition of 2 vol(O.2 ml) of magnetic beads (in bead prehybridization buffer) containing approximately 10 pmol of bound oligo(dT). The procedure was repeated for the labeled probe at the optimized capture probe concentration.

Determination of the Optimum Concentration

Magnetic Bead

The appropriate concentration of oligo(dT) magnetic particles to use depends on many factors. To determine the optimal concentration of beads to use in an assay, beads were concentrated to a dA12 binding capacity of 3 nmol/ml in prehybridization buffer and serially diluted into prehybridization buffer. Once again, two hybridizations were prepared. Each hybridization contained 1.0 ml. The first or experimental sample generally included about 10 fmol of target (with or without a biological matrix), 1.0 pmol (or other optimized value) of labeled probe, and 50 pmol (or other optimized value) of the tailed capture probe. The second or control sample was identical to the first except that a component was omitted (either the target or the capture probe) or a nonhomologous tailed probe or preferably a nonhomologous target was used. The experimental and control samples (0.1 ml) were mixed with 0.2 ml of the serial dilutions of beads and incubated for 5 min at 37’C. The final GuSCN concentration during capture was 0.83 M. The beads were washed three times and counted. The signal/noise ratio was plotted against bead dilution and the concentration of beads giving the highest signal/noise ratio was chosen.

d&TAILED

CAPTURE

Reversible Target Capture Assay. Dose Response: Detection of Serial Dilutions of Listeria Monocytogenes in the Presence of 1.25 X 10’ Campylobacter The following assay format used all of the optimized parameters for the detection of Listeria 16 S rRNA with RTC. The composition of the special RTC buffers used in this procedure may be found in Table 1. Samples (0.25 ml) were made 45 nM (0.50 pg/ml) in the Listeria-specific capture probe (No. 773-dAiZs) and approximately 1.0 nM in the 32P-labeled eubacterial riboprobe (0.27 pg/ml). The final concentration of GuSCN in the hybridization mixture was 2.5 M. The hybridizations also contained (final concentrations) 0.2 M TrisHCl (pH 7.5), 0.04 M EDTA, 0.5% Sarkosyl, and 10% dextran sulfate-5000, C. jejuni cells (lO’/ml), and serial dilutions of Listeria cells. Three controls were performed containing either no Listeria, no tailed probe, or no cells (no Listeria or Campylobacter). Samples were incubated for 15 min at 37OC. The 0.25ml samples were mixed with 0.5 ml of magnetic beads (in bead prehybridization buffer; Table 1) containing 62.5 pmol of oligo(dT) (five- to sixfold molar excess). The samples were vortexed vigorously for 10 s and capture was allowed to proceed for 5 min at 37’C in 12 X 75-mm polypropylene tubes. The GuSCN concentration during capture was thus 0.83 M. The tubes were placed in a magnetic separator (Corning) until the solution was visibly cleared of beads. The liquid was then either decanted or removed with a transfer pipet. Beads were resuspended in 0.6 ml of bead wash buffer (Table l), vortexed for 10 s, and magnetically separated. The beads were washed twice more (three washes total). As much as possible of the remaining fluid was removed. Prewarmed (37OC) chemical elution buffer (0.25 ml; Table 1) was added and the samples were vortexed vigorously and incubated at 37OC for 2 min to elute intact ternary complexes. Samples were magnetically separated, and the fluid was withdrawn into fresh tubes containing 0.5 ml of magnetic beads (containing 62.5 pmol of oligo(dT). The beads were washed twice as above. Prewarmed thermal elution buffer (0.5 ml; Table 1) was then added, and the samples were vortexed vigorously and incubated for 2 min at 68.5OC. The samples were prefiltered through preblocked polysulfone or Teflon acrodiscs (Gelman, 0.2 pm). At this point the samples were split into two for the purposes of comparing a triple bead capture with a double bead filter capture. One-half (0.25 ml) of each sample was recaptured on a third set of beads (in 0.5 ml prehybridization buffer plus 1.0 M NaCl) at 37’C. The other half (0.25 ml) of each sample was slowly filtered through the preblocked poly(dT)*OOO nylon at room temperature (approximately 0.05 ml/min) to concentrate the nucleic acid targets. (Polysulfone and Teflon prefilters were pre-

PROBES.

349

I

blocked as described previously for nylon filters.) Filters or beads were washed and the amount of label associated with the target was measured by scintillation counting (beads or filters) or autoradiography (filters). RESULTS

Fundamentals

of Reversible Target Capture

The process of RTC is illustrated in Fig. 1. A single ternary complex is highlighted in the box of part 1. This is the signal-generating entity that is formed by two hybridization events: that between the target and the labeled probe (wavy line with dark circles) and that between the target and the dA-tailed capture probe (wavy line with the dAs). Surrounding the ternary complex are many unhybridized labeled and tailed probes. The assay uses a vast molar excess of probe over the target in order to drive the hybridization to completion in 15 min or less. However, the unhybridized labeled probes constitute noise unless they are removed prior to detection. RTC was designed to eliminate this noise. In part 2 of Fig. 1, the ternary complex has been captured onto an oligo(dT) magnetic bead by means of a hybridization between oligo(dT) and poly(dA). Note, however, that there are numerous labeled probes that are nonspecifically bound to the surface of the bead as well (between 0.01 and 0.1% of input probe). By “nonspecifically bound” is meant “bound in the absence of a target molecule.” Nonspecifically bound background is generally denoted simply as NSB. In the case pictured, there would be five labeled probes bound nonspecifically for every one bound specifically if the assay was stopped at this point. This would make it difficult to quantify the target present in the initial sample by this single capture method. The beads are washed well to reduce as much NSB as possible, but some background is resistant to washes. The system is designed so that the weakest hybrid in the captured target is the one between poly(dA) and oligo(dT). Thus “elution” conditions can be applied in which the hybrids between poly(dA) and oligo(dT) are selectively broken, leaving the ternary complex intact. The first set of beads with some of the initial NSB is then discarded. The intact ternary complex is then recaptured on another set of beads. Most of the NSB (usually >99%) that bound in round 1 has been removed. However, the reduction in NSB is still not sufficient for quantitating subattomole levels of target in highly impure specimens. Thus the process is repeated until the noise is either low enough to achieve the desired sensitivity or even undetectable as in part 3 of Fig. 1, where the ternary complex is shown bound to the final or detection solid phase and there is no detectable NSB. Generally, on the final solid phase, the oligo(dT) is replaced by poly(dT)dOOO so that stringency washes can be performed (if desired) with minimal disruption to the poly(dA-poly(dT) hybrid

350

MORRISSEY REVERSIBLE

TARGET CAPTURE.

UNHYBRIDIZED

PART 1.

LABELED PROBES

TERNARY COMPLEX

(NOISE)

(SIGNAL)

--y-

PART 2.

NONSPECWMLY USELED PRO&s

RWERSIELE

TARGET CAPTURE.

AL.

that holds the entire ternary port.

complex to the solid sup-

The Binding of Poly(dA) and Poly(rA) to Oligo(dT)14 as a Function of the Concentration of GuSCN 5’

RWERSIBLE TARGET CAPTURE.

ET

0OUNrJ (NOISE)

PART 3

A key element to the successful use of RTC is to design the system such that the hybrids in the ternary complex are stronger than the hybrid between poly(dA) and oligo(dT). To accomplish this, the length of the oligo(dT) can be adjusted to suit the oligonucleotide capture probes being used. In many cases the length and GC composition of the oligonucleotide capture probe can also be adjusted. The stability of poly(dA)-oligo(dT)14 was examined in detail at 37’C at various concentrations of GuSCN in Appendix A. The salient points are summarized below. At GuSCN concentrations above 1.0 M, the binding of poly(dA) to oligo(dT) begins to decrease. Thus the concentration of GuSCN in the binding and wash buffer should not exceed 1.0 M at 37OC. At GuSCN concentrations above 2.0 M, the poly(dA)-oligo(dT) hybrid is virtually completely dissociated. Thus GuSCN concentrations 2.0 M and higher may be used as elution buffers at 37OC. The upper limit to the GuSCN concentration that can be used for elution is determined by the stability of the ternary complex. If the ternary complex dissociates in the elution buffer, it must be reformed prior to capture. Since this rehybridization is considerably slower than recapture, it is desirable to avoid destabilizing the ternary complexes. Guidelines for Probe Selection

EL-J POLY(dT)

FILTER

dT

FIG. 1. Schematic diagram of reversible target capture (RTC). Part 1. A ternary complex consisting of the target, the capture probe (wavy line with the dAs), and the labeled probe (wavy line with dark circles) is highlighted in the box. Surrounding the ternary complex are many unhybridized labeled probes, which constitute assay noise. RTC was designed to eliminate this noise, thus permitting very fast hybridixation with high sensitivity. Part 2. The ternary complex has been captured onto an oligo(dT) magnetic bead by means of a hybridization between oligo(dT) andpoly(dA). Note, however, that there are numerous labeled probes that are nonspecifically bound to the surface of the bead as well. The beads are washed well to reduce as much NSB as possible, but some background is resistant to washes. The system is designed so that the weakest hybrid in the captured target is the one between poly(dA) and oligo(dT). Thus “elution” conditions can be applied in which the hybrids between poly(dA) and oligo(dT) are selectively broken, leaving the ternary complex intact. The first set of beads

As mentioned above, successful use of RTC depends on the complex of ternary capture probe-target-labeled probe having a greater stability than that of oligo(dT)poly(dA) in capture buffers, wash buffers, stringency washes, and elution buffers. The stability of the ternary complex depends on the less stable component, which is ideally the hybrid between the capture probe and the target. The labeled probe-target hybrid should be stronger than the capture probe-target hybrid because the labeled probe-target hybrid has to survive a drastic reduction in the molarity of the labeled probe during triple capture whereas there is excess capture probe in the system. Figure 2 summarizes the most important points. The labeled probe can be either a 3’- or 5’-labeled oligonucleotide for optimal specificity of hybridization or

with some of the initial NSB is then discarded. Because the ternary complex remains intact, rapid capture/release cycles can be performed repeatedly until the noise is low enough to achieve the desired sensitivity. Part 3. The target has been captured on the final solid phase for detection. The noise is now undetectable, allowing very faint signals to be quantified accurately.

dATAILED

CAPTURE

GUIDELINES FOR PROBE SELECTION

PREFERRED IABELED PROBES: 1. RIBOPROBE. 2 Preferred

isotopic

Preferred

nonisotopic

PREFERRED CAPTURE PROBE:

Optimal

Probe

Length:

Td of oligo-target GC content dA-tail

1. ‘2P.

label:

2.

PROEE.

‘*‘I.

Biotic.

OLIGONUCLEOTIDE.

Td of 35mer. Td of 35mer.

Optimal

labels:

NICK-TRANSLATED

30-40

should

in 2.5 M GuSCN. in 2.5 M GuSCN.

be at least

= 100-200

nucleotides.

FIG. 2.

Guidelines for probe selection. The labeled probe should form a tighter hybrid with the target than the capture probe. In GuSCN, RNA double helices are the strongest hybrids. In addition, riboprobes have the lowest NSB on magnetic beads. Thus they are the preferred labeled probes. Oligonucleotides are the preferred capture probes because of their greater specificity. The strength of the oligonucleotide capture probe-target hybrid must exceed that of the oligo(dT)-poly(dA) hybrid in order to perform rapid cycles of elution/ recapture with intact ternary hybrids. Recommended dissociation temperatures in 2.5 M GuSCN (Td’s) of oligonucleotide-target hybrids are greater than 42’C with oligo(dT)14 beads and greater than 27’C with oligo(dT)10 beads. Alternatively, with short, AT-rich oligonucleotides, substitute 0.5 M GuSCN or 0.5 M NaCl for 2.5 M GuSCN for hybridization and substitute thermal elution for chemical elution in 2.5 M GuSCN.

preferably a riboprobe for optimal detection sensitivity. Nick-translated probes have also been used for detection of subattomole quantities of target (25). Use of biotinylated probes in RTC is described in detail in the accompanying paper (25). Oligonucleotides are the preferred capture probes because of their greater specificity (26,27). Standard hybridization conditions are 37°C and 2.5 M GuSCN. This is also the standard condition for chemical elution. A dissociation temperature of the oligonucleotide capture probe-target hybrid of 42’C or greater in 2.5 M GuSCN is recommended. Generally, oligonucleotide capture probes 30-40 nucleotides long will meet this criterion. GC content greater than 50% is preferred since the dissociation temperatures of oligomers in GuSCN are strongly influenced by GC content. For example, in 2.5 M GuSCN, a 35-mer with 37% GC dissociates at 39OC, while a 35-mer with 66% GC dissociates at 53OC (data not shown). Determination Temperature

ternary complex, which is designed to be the capture probe-target hybrid. The method for determining the optimal salt and temperature has been explained in detail under Materials and Methods. The standard hybridization conditions of 37’C in 2.5 M GuSCN have been found to be optimal for hybridization to L. monocytogenes 16 S rRNA with the 35-mer oligo probe No. 773 (57% GC), which is used to illustrate RTC below. Determination of the Optimal Capture and Labeled Probe Concentrations

42’ in 2.5 M GuSCN.

> 5OZ preferable. length

351

I

nucleotides.

37% GC = 39 degrees 66Z GC = 53 degrees duplex

PROBES,

of the Optimum Hybridization and Salt Concentration

The optimal hybridization temperature and salt depend on the relative strength of the secondary structures in the target region and the least stable hybrid in the

The optimal capture probe concentration depends on the time of hybridization and on the degree of secondary structure in the target regions. Generally, oligonucleotide capture probes are used between 1 and 50 nM (depending on the target) in 2.5 M GuSCN at 37’C for a 15min hybridization. To determine the optimal capture probe concentration, the optimal temperature and salt concentration for the hybridization must be known. As noted above, optimal hybridization of L. monocytogenes 16 S rRNA with the 35-mer Listeria-specific probe No. 773 occurs under standard conditions of 2.5 M GuSCN and 37OC. The experiment is explained in detail under Materials and Methods and is outlined in Fig. 3. The basic protocol of Fig, 3 was followed to determine the optimal capture probe concentration for probe No. OPTIMIZING

CAPTURE

1. Determine

PROBE

optimal

and L4BELED

temperature

PROBE CONCENTRATIONS

and salt first.

2. Set up 2 sets of hybridization rewtions. Suggested volume 0. Variable capture probe, fixed target, fixed labeled probe. b. Variable capture probe, no target, fixed labeled probe. Recommended levels. Use 0.1 picomole of ‘2P-lobeled riboprobe Use dA-toiled capture probe between 0 I Use 1 femtomole of target. 3. Suggested

time of hybridization

= 0.1 ml.

(106 cpm/ug), and 5 pmoles.

= 15 min.

4. Suggested salt and temperotwe 0s determined previously (Hybridize at 15 degrees below Td of least stable probe) 5. Add 0.2 ml of oligo(dT) beads containing 10 pmoles of oligo(dT). Incubate for 5 min at 37 degrees for capture. (GuSCN) during capture should be M.

0.8-1.0

6. Wash beads 3 times and ecintillotion

count.

7. Repeat for labeled probe using optimized

FIG. 3.

capture

probe.

Procedure for optimizing probe concentrations. An outline of the most important features is given here. The optimal probe concentration is that which maximizes the signal/noise ratio of the hybridization assay. With the standard hybridization conditions of 2.5 M GuSCN, 10% dextran sulfate at 37’C a 15-min hybridization has a maximum signal/noise ratio with the capture probe between 1 and 50 nM, depending on the structure of the target region. Whenever possible, target sites with minimal or weak secondary structures should be chosen. Generally, labeled riboprobe has a signal/noise optimum of approximately 1.0 nM (i.e., about 0.27 pg/ml for a 770-mer probe) under the standard conditions.

352

MORRISSEY

(Capture

Probe),

nM

FIG.

4. Optimization of the L&e&-specific capture probe (No. 773) concentration. The signal (circles) and noise (triangles) in counts per minute are displayed against the concentration of capture probe in the hybridization mixtures. The 3 X 104 Listeria cells (1 fmol of target) or an equivalent number of E. coli cells (control) were incubated with the indicated concentrations of Listeriu-specific capture probe and 1 nM labeled generic “P-labeled riboprobe for 15 min at 37OC in buffered 2.5 M GuSCN containing 10% dextran sulfate. The O.l-ml samples were mixed with 0.2 ml of beads containing 12.5 pmol of oligo(dT) and capture was allowed to occur for 5 min at 37’C. The beads were washed twice and counted.

773, except for slight deviations in the suggested capture probe concentrations. The results are shown in Fig. 4. Backgrounds in this case are equivalent at all capture probe concentrations (510 k 60 cpm). The optimal capture probe concentration is thus determined by the maximum signal in this case. For the 35-mer probe No. 773 the optimal capture probe concentration is between approximately 33 and 66 nM in 2.5 M GuSCN at 37OC. At a probe concentration of 70 nM, 0.1 ml contains 7 pmol. At higher probe concentrations, the signal falls off rapidly as the amount of capture probe in the sample approaches and then exceeds the amount of oligo(dT) (12.5 pmol). At lower probe concentrations, there is a gradual decline in captured signal due to insufficient probe to drive the hybridization to completion in 15 min. Labeled probe concentration can be similarly optimized using the optimal salt, temperature, and capture probe concentration. Determination of the Optimal iMagnetic Bead Concentration The optimal magnetic bead concentration depends in a complex fashion on many factors, including the time alloted for capture, the capture probe concentration, the target size (and most likely the target tertiary structure), the chemical composition, the temperature, the viscosity of the solvent used to perform target capture, the dissociation temperature of the oligo(dT)-poly(dA) hybrid in the capture solvent, the number of capture probes per target, and also the dA tail length. The optimal level to

ET

AL.

use is always that which provides the highest assay signal/noise ratio. Since the signal/noise ratio can change rapidly with changes in the bead concentration, it is best to optimize for the target of interest with the simple experiment described below. Generally, a 5- to lo-fold molar excess of oligo(dT) over poly(dA)-tailed probes provides maximum signal. Once the optimal capture probe concentration is determined, the optimal bead concentration is readily determined. The standard procedure is described in detail under Materials and Methods. In the experiment described below the volumes in the standard procedure were reduced by a factor of two and the amount of target was increased lo-fold. These changes have no effect on the nature of these curves (data not shown). Here the method is applied to determine the optimal concentration of oligo(dT) beads for capture of Listeria 16 S rRNA. Having determined that approximately 50 nM oligo capture probe is optimal for capture of 16 S Listeria rRNA with a 15-min hybridization in 2.5 M GuSCN at 37’C, it remained to be determined whether improved results could be achieved with a different concentration of magnetic beads. For this experiment two 0.5-ml hybridizations were prepared. One contained 100 fmol of Listeria rRNA and the other contained an equivalent amount of E. coli rRNA as a control. Both mixtures contained approximately 45 nM oligo capture probe and 1 nM “P-labeled generic riboprobe, in buffered 2.5 M GuSCN, 10% dextran sulfate. Hybridization was for 15 min at 37OC. At this point 0.05 ml (containing 2.25 pmol of capture probe and 10 fmol of target) was withdrawn from each mixture and added to 0.1 ml of magnetic beads containing between 2.44 (0.3 mg/ml solids) and 312 (40 mg/ml solids) pmol of dTid. The capture was performed for 5 min and the beads were washed twice and scintillation counted. The results are shown in Fig. 5. In Fig. 5A note that there is a loss in signal between 78 and 312 pmol of dTid. The reason for this is unknown. There is a broad signal optimum, followed by a significant (2.5-fold) signal loss at 4.88 pmol of dAiZ binding capacity. Theoretically, this is still almost twice the number required to bind the 2.25 pmol of added capture probe. However, control experiments have shown that the signal obtained with this dilution of beads does not increase even after 30 min of capture (data not shown). This indicates that it is not simply a kinetic problem. Rather, there is insufficient capacity. Perhaps the dAiZ capacity slightly overestimates the true capacity for tailed probe-target complexes in that because the latter is much larger, some sites available to bind dAiZ may be unable to bind high molecular weight ternary complexes. The nonspecific noise decreases monotonically (although not linearly) with bead dilution (Fig. 5B). As a result of these data, there is an optimum bead concentration window between approximately 9.75 and 19.5 pmol

d&TAILED 200-

.ze 07-

CAPTURE

A.

loo-

50-

312

156

78

33

Picomoles

I9 5 dT,4

3 ?5

4.88

244

Added

FIG. 5. (A) The signal obtained with &sterk rRNA at various bead dilutions. The standard procedure is described in detail under Materials and Methods. Two minor changes were made to the standard procedure: Volumes were scaled down twofold and 10 times more target was used. A 0.5-ml hybridization was set up containing 100 fmol of Wisteria rRNA (from crude cell extracts), 22.5 pmol of dA-tailed capture probe, 0.5 pmol of “P-labeled riboprobe (3.1 X 10m5 cpm/molecule), buffered 2.5 M GuSCN, 10% dextran sulfate. After a 15-min hybridization at 37C, 0.05 ml-ahquots of the hybridization mixture were mixed with 0.1 ml of the bead dilutions containing the indicated number of picomoles of oligo(dT)i4. The beads were washed three times and counted. (B) The noise obtained with E. cok at various bead dilutions. A hybridization was set up as in A except that 100 fmol of E. co& rRNA (from crude cell extracts) was used in a 0.5-ml hybridization as a control. (C) The signal-to-noise ratio at various bead dilutions. The signal of A was divided hy the noise of B at each of the indicated bead dilutions.

of dTId ligand (Fig. 5C). This is a 4.3- to 8.7-fold excess over the tailed oligonucleotide probe.

molar

Illustration of the RTC Procedure with a Dose-Response Curve: Detection of Six Listeria Cells in the Presence of 1.25 X 1O7Campylobacter Cells Now that all of the parameters have been optimized for the capture of 16 S rRNA from Listeria, the rest of the paper will analyze hybridization data generated by RTC as a function of the number of rounds of capture. The experimental protocol for the dose-response study is described in detail under Materials and Methods. Briefly, 0.25-ml samples containing 2.5 X 107 Campylobatter control cells were made 45 nM (0.5 pg/ml) in the Listeria-specific capture probe (NO. 773~dA& and ap-

PROBES,

I

353

proximately 1.0 nM in 32P-labeled generic eubacterial riboprobe (0.27 pg/ml). The final concentration of GuSCN in the hybridization mixture was 2.5 M. Samples were incubated for 15 min at 37’C. The O-25-ml samples were mixed with 0.5 ml of magnetic beads (in bead prehybridization buffer; Table 1) containing 62.5 pmol of oligo(dT) (>fivefold molar excess), and capture was allowed to proceed for 5 min at 37’C in 12 X 75-mm polypropylene tubes. The beads were washed three times as described with 0.6 ml of bead wash buffer (Table 1). At this point, as much as possible of the remaining fluid was removed. Prewarmed (37OC) chemical elution buffer (0.25 ml; Table 1) was added and the samples were vortexed vigorously and incubated at 37’C for 2 min to elute intact ternary complexes. The fluid was withdrawn into fresh tubes containing 0.5 ml of magnetic beads (containing 62.5 pmol of oligo(dT)). The beads were washed twice as above. Prewarmed thermal elution buffer (0.5 ml; Table 1) was then added and the samples were incubated for 2 min at 68.5’C. After the samples were prefiltered through Teflon acrodiscs, they were split into two for the purposes of comparing a triple bead capture with a double bead filter capture. One-half (0.25 ml) of each sample was recaptured on a third set of beads (in 0.5 ml prehybridization buffer plus 1.0 M NaCl) at 37C. The other half (0.25 ml) of each sample was slowly filtered through the preblocked poly(dT)dOO,, nylon at room temperature (approximately 0.05 ml/min) to concentrate the nucleic acid targets. Filters or beads were washed and the amount of label associated with target was measured by scintillation counting (beads or filters) or autoradiography (filters). Illustration of the RTC Procedure. (A,l The Effect of Multiple Rounds of Capture on the Signal of the Hybridization Assay The effect of multiple rounds of capture on the size of the signal of the hybridization assay was determined in the assay format just described. All beads, filters, and samples that did not bind to the beads were counted, Washes amount to less than 5% of what did not bind to the beads and were discarded. From these raw data the formulas of Table 2 were used to calculate the signal after each round of capture. From the measured signal, the known specific activity of the riboprobe (2.4 X 10m4cpm/ molecule), and the number of targets added to the hybridization, the efficiency of capture was calculated for each round. The results are shown in Table 3. After three captures, there is approximately one-half of the target that was present after round one. The bead capture efficiencies were approximately 55-95% per round and the filter capture efficiencies were about 75%. The efficiency is independent of target concentration (see below also). The

354

MORRISSEY TABLE Formulas

for

Calculating of Round

TABLE

Signal Number

as

a Function

Types

+ 2 *B3

Note. Bl, counts per minute left on the first set of beads. DNRB2, counts per minute that did not rebind to second set of beads. B2, counts per minute left on the second set of beads. DNRB3, counts per minute that did not rebind to the third set of beads. B3, counts per minute on the third set of beads. Filter, counts per minute captured on the nylon filter.

overall efhciency of the assay is the product of the efhciency of each round. The efhciency of capture in the first round is the product of the efficiency of the hybridization of the capture probe (&,) to the target, the efficiency of the hybridization of the labeled probe (HJ to the target, and the efficiency of the capture of the target onto the beads (HJ. The efficiency of one cycle of elution/recapture is the product of the efficiency of elution (EJ and the efficiency of recapture (IQ. Thus the overall efficiency of capture (EJ of the capture probe-targetlabeled probe ternary complex on the third solid support may be written as = HcP*Hl*H,,*(Ee*E&*(Ee*EJ.

The overall efficiency of the process is the product of seven factors. For an overall efficiency of 45%, e.g., the average efficiency per step is thus about (0.45)li7 or about 90%. Ten to thirty percent overall efficiency is typical for RTC for three rounds of capture. If the overall efficiency is less than lo%, the assay needs to be reoptimized.

TABLE Efficiency

of Target

Number of target cells

100,000 10,000

Capture Cumulative after

1

2

77 96

43 56

Note. The dose-response text. From the formulas of samples containing 100,000 calculated. From the known X lo-’ cpm/molecule) and cell (20,000), the cumulative capture.

AL.

2

First round data points = Bl + DNRB2 + B2 + 2 *DNRB3 Second round data points = B2 + 2 * DNRB3 + 2 * B3 Third round data points = 2 * B3 or 2 * filter

E,, = El*E2*E3

ET

3 after

Each

Round

efficiency of capture round number 3 (bead) 36 49

3 (filter) 31 44

study was performed as described in Table 2, the net amount of signal in Listeriu cells and 10,000 Gsrer~o cells specific activity of the riboprobe the known number of target rRNAs efficiency is displayed after each round

the the was (2.4 per of

Control number

Target cells: Listeria

Cl c2 c3 Note.

of Controls

for

Heterologous Campylobacter

No No No These

controls

4

Dose-Response

Experiments cells: Tailed

Yes Yes No are defined

in detail

probe

773

Yes No Yes in the text.

Illustration of the RTC Procedure. {BJ The E#ect Multiple Rounds of Capture on the Noise of the Hybridization Assay

of

In the dose-response experiment described above three controls that are defined in Table 4 were performed to measure assay background. There are dozens of useful controls that can be performed with RTC to identify precisely the sources of the assay background. Control Cl (heterologous target) measures the hybridization-dependent background (NSH) plus the hybridization-independent (NSB) background. Control C2 (no tailed probe) measures the hybridization-independent background (NSB). Control C3 (no cells) measures the NSB of the labeled probe to the supports plus the background caused by the interaction of the capture probe and the labeled probe. Control Cl minus control C2 measures the hybridization-dependent background, which comes from misformed hybrids between the capture probe and the closely related nucleic acids. This type of background is controlled by stringency washes and by introducing additional levels of specificity. Control C2 minus control C3 measures the increase in assay background caused by the binding of cellular components to labeled probe. Cellular fractionation can be used to identify and eliminate these components. Table 5 shows a typical profile for background reduction for the reversible target capture procedure. The noise is that of control Cl in Table 4: No target cells are present. Capture probe, 3.1 X 107 cpm of labeled probe, and heterologous cells were incubated with the indicated solid supports in the absence of the target to measure the level of nonspecific background of the assay. At the end of the first round of capture the nonspecific background was reduced approximately 4200fold (3.6 logs). Each succeeding round of capture reduces the background by less than the previous round (suggesting an asymptotic approach to the a limiting background). The second round of capture removed an additional 2.1 logs of background. The third round of bead capture removed another 1.3 logs of nonspecific background. When the third round of capture was performed on a filter instead of on magnetic beads, there was a 1.9 log reduction in background. In summary, then, triple bead capture re-

dA-TAILED CAPTURE PROBES, I TABLE 5 Reduction of Assay Noise after Each Round of Target Capture Total Round

number

noise

3.1 x lo7 7.4 x lo3

Zero One

5.8 x lOI

Two Three Three

assay Cwnl

(bead) (filter)

3.3 0.7

Log reduction assay noise (cumulative)

in

N.A. 3.6 5.7 7.0 7.6

&‘o&. The absolute value of the noise in counts per minute (control Cl, no L&eria) is displayed by round of capture during the dose-response experiment. Round zero refers to the amount of labeled probe added to the samples prior to the first capture. At the right, the logarithm of the cumulative noise reduction is displayed by round for the dose-response experiment. The formulas of Table 2 were used to calculate the counts per minute associated with the Cl control after each round. The Cl background on the final filter was calculated by comparing the relative intensities of the Cl autoradiographic band to that of reference bands (containing at least 20 cpm) that were both scanned and counted in scintillation fluid. N.A., not applicable.

cluced the background by 7 orders of magnitude and double bead/filter capture reduced the background by a total of 7.6 logs.

355

(Ti) (a plot of molecules of ternary complex captured against initial target number is a straight line with a slope equal to Z&). This implies the validity of formula [7] of Appendix B between 6 and 50,000 Listeria. Since the slope of the linear dose-response curve is 1.0, reasonably precise quantitation of the target in a sample between 6 and 50,000 Listeria cells (between 120,000 and 10’ target molecules) was achieved. The line at the bottom of the curve represents the average background (control Cl). Figure 7 shows the advantage of triple capture over single capture methods by displaying the improvement in detection limit after each round of bead capture. At the end of round one, 10,000 Listeria cells produced a signal (about 43,700-7400 cpm) that was about 5 times background (7400 cpm). From formula [7] (Appendix B) the detection limit at the end of the first round is thus about 5000 Listeria cells. These results are very similar to the detection limit seen previously (14) after one round of target capture on polystyrene: 12,000 SalmonelZa (which also contain about 20,000 ribosomes/cell) were detected at 10 times the background. At the end of the second round of bead capture, the signal from 333 Listeriu (1156-130 cpm) is visible at about eight times background (conservatively, 130 cpm is taken as background because it is anomalously higher

Illustration of RTC. (C) Dose Response after One, Two, and Three Rounds of Capture on Magnetic Beads The data of the previous sections showed that the signal declined only about two- to threefold during triple capture (triple bead and double bead filter) while the nonspecific noise declined by 7-8 orders of magnitude during triple capture. Triple capture thus enhances the signal-to-noise ratio by 6.5-7.5 orders of magnitude. Considerations presented in Appendix B predict that as few as six Listeria cells should be detectable at 5.5 times background with this assay format, given 1.3 X 10” labeled probes input, 120,000 initial targets (equivalent to six cells), 7.3 logs of background reduction (average of triple bead and double bead filter captures), and 30% overall efficiency of capture of the target. Data are presented below that show how close this assay comes to its predicted performance. Figure 6 shows the dose-response curve between 6 and 50,000 Listeria cells after three rounds of bead capture. Log (signal) was plotted against log (cell number) in order to display all of the data points clearly on the graph. The data from two independent experiments were averaged to make this plot. The dose response is linear over this 4 log range of target and its slope is equal to 1.0. The linearity of the dose-response curve implies that the overall efficiency of capture (EJ is independent of initial target number

FIG. 6. Dose response between 6 and 50,000 L&e&z: triple bead capture. This dose-response experiment is described in detail in the text. Listeriu cells were serially diluted into 10s Carnpylobacter cells/ ml in the standard 2.5 M GuSCN hybridization cocktail. The sample volume was 0.25 ml. The Lkterm-specific capture probe No. 773-dAne was used at 45 nM and the “P-labeled riboprobe (2.4 X 10m4 cpm/molecule) was added at 0.86 nM. After three rounds of capture on oligo(dT) magnetic beads, log(signa1) was plotted against the log of the number of Listwiu cells initially added to the hybridization mixture. The line at the bottom of the graph is the log of the counts per minute associated with control Cl of Table 3. The data from two independent experiments have been averaged to make this graph.

356

MORRISSEY 4001

A.

One Round

of Capture

I

ET

AL.

cisely (even after 100 min of counting) the signalto-noise ratio (S/N) on which the detection limit (D) depends (formula [5] Appendix B) because of the relatively high.machine background (30-40 cpm). For this reason a final capture on a poly(dT) filter is preferred for this type of measurement since extremely low signals and backgrounds can be measured very precisely using X-ray film, as discussed below.

Illustration of RTC. CD) Dose Response after Two Rounds of Capture on Magnetic Beads and a Final Filter Capture A double bead/filter capture scheme allows a much more precise determination of the S/N ratio of the hybridization assays at low target number. Figure 8A shows a typical autoradiogram for the detection of between 6 and 56 Listeria with double bead/filter capture. The densitometric trace of the least intense bands of this typical double bead/filter capture is shown in Fig. 8B directly below its respective band. Peak “C3” in Fig. 8B is the background that contained no cell extract. Typically, this is somewhat lower than controls that contain cell extracts. It measures both the interaction between the two probes and the pure NSB of Number

of Listeria

FIG. 7. (A) Dose response after one round of bead capture. The dose-response experiment described in detail in the text is displayed as a function of the round of capture. This most clearly illustrates the advantages of reversible target capture. The counts per minute bound to the beads at the end of the first round (just prior to the first elution) were calculated from the formulas of Table 2 and plotted for each indicated number of Lkteriu cells. The three backgrounds denoted 0 L&er&a cells in the figure are as described in Table 3. These controls are left to righti control 1, no Listeriu; control 2, no tailed probe; and control 3, no cells. (B) Dose response after two rounds of bead capture. The samples in A were incubated with 0,25 ml of chemical elution buffer at 3YC for 5 min and recaptured with the addition of 0.5 ml of fresh beads (in bead prehybridization buffer). The formulas of Table 2 were used to estimate the signal after two rounds of capture. (C) Dose response after three rounds of bead capture. The samples in B were incubated with 0.5 ml thermal elution buffer at 68.5OC and 0.25 ml of each of the samples was recaptured on a third set of beads. Accordingly, the number of L&erti cells pictured here is one-half of the number pictured above. The data presented here are the lOO-min scintillation counts of the third set of beads.

than control Cl, which has 58 cpm of background). From formula [7] of Appendix B, the detection limit has improved to about 125 cells. Note that 111 cells is 2.3X background. Thus one would predict that the detection limit is just slightly higher than 111 cells. At the end of the third round of beadcapture, six Listeriu give a signal (S-3 cpm) that is four times background (3 cpm). Thus approximately six Listeriu cells are the detection limit. At this low a target number (approximately 120,000 total), it is difficult to measure pre-

B.z.“m I6

!

14

’

II I

04.

00 bJ4

-02117.7

I50 0

FIG. 8. (A) Autoradiogram of double bead/filter capture. After two rounds of capture on beads, the samples were treated with thermal elution buffer to release targets. A final capture was performed on a preblockedpoly(dT) GeneScreen filter. Filters were washed bypassing 0.1 ml of prehybridization buffer through each slot in the apparatus. The poly(dT) nylon filter was dried under an infrared lamp and exposed to Kodak XAR film for 3.5 days at -&WC with two intensifying screens. The band designations refer to controls C3, C2, and Cl (Table 4) and 6,19, and 56 L&term. (B) Shimadzu CS930 densitometric trace of the autoradiogram of A. A densitometric trace is shown directly under the four least intense bands.

dA-TAILED

CAPTURE

the labeled probe to the solid phases in the experiment. Both numbers are values in the absence of cells. The presence of one or more components in cells increases the NSB of the labeled probe to the supports (peaks “C2” and “Cl”). Note that peaks C2 and Cl in Fig. 8B are the same size. This indicates that there is no NSH between the Listeria capture probe No. 773 and Campylobacter rRNA, since the only difference between the controls is the presence or absence of the capture probe. Thus Campylobacter rRNA is not a source of background. This is expected since the Listeria-specific probe sequence was chosen because it is very divergent from other eubacteria in this region (Dr. David Lane, data not shown). Presumably, this also means that with highly purified RNA, the assay background would be that of peak C3 and the assay detection limit would be improved by a factor of about five. These controls can be valuable in measuring the relative contributions of NSB and NSH to the total background. Also, by separating the cell extracts into fractions, it is possible to determine the sources of the cellular backgrounds. Control experiments have shown that cellular proteins are usually the major source of NSB (data not shown). Peak “6” is the signal from six Listeria. Its area, minus background (37,735 - 7000), is 4.4 times the area of trace No. 3 (7000). Thus the detection limit is approximately six Listeria cells (formula [6] of Appendix B). In a repeat of this experiment six Listeria were detected at 8.4 times background (data not shown). This is close to what is predicted by the formulas of Appendix B, namely that six Listeria cells should be readily detectable at about 5.5 times background. DISCUSSION

A hybridization assay called RTC has been developed that is rapid (about 1 h for triple capture) as well as quantitative. A series of quick experiments are performed to optimize salt and temperature of hybridization, as well as the appropriate concentrations of capture probe, Iabeled probe, and magnetic beads. Standard conditions have been defined for 30- to 40-mer oligonucleotide capture probes and labeled riboprobes. The method involves releasing nucleic acids from cells and hybridizing a dA-tailed capture probe and one or more labeled probes to the target molecule in solution. The capture probe-target-labeled probe ternary complex is then rapidly captured onto an oligo(dT) support. The support is washed and the target released. The target is rebound to a fresh oligo(dT) support, washed, and released. It is recaptured onto oligo(dT) beads or onto poly(dT) nylon filters for detection with either scintillation counting or autoradiography. After one round of capture, excess labeled probe is reduced by about 3.6 orders of magnitude and the detection limit is between 5 X 107 and 2 X 1O’target molecules.

PROBES,

I

357

After an additional round of capture, excess labeled probe is reduced by another 2.1 orders of magnitude and the detection limit improves to about 2 X 106 molecules. After a third round of capture, the excess labeled probe is reduced another 1.3-1.9 orders of magnitude and the detection limit improves to approximately 4 X 104 to 1 X 105 molecules. The intent of using multiple solid phases with different surface chemistries is to remove backgrounds caused by the presence of a class of molecule with a high atllnity for a particular solid phase. Exactly analogous arguments explain the use of very different types of elution conditions (chemical, thermal, and even enzymatic). The decrease in the degree of background reduction per round from 3.6 in the first round to only 1.3-1.9 in the third round, however, suggests an approach to an asymptotic or limiting background. This limiting background could in some cases be a very low level of target or a class of molecule (e.g., labeled probes complexed with some type of cellular macromolecule) with high affinity for two very different solid phases (magnetic particles and nylon filters) and a tendency to elute from beads with both chemical and thermal elution buffers. When fully optimized, the average efficiency of capture (recapture) is about 65-75% per round. It is therefore tempting to push RTC a bit further to see if four or even five captures can significantly improve the sensitivity below 0.2 amol (this paper). In the accompanying paper (25), the sensitivity is indeed improved to less than 0.01 amol with a fourth target capture. Limitations to sensitivity caused by the imperfect specificity of the probes can be overcome as described (14,25). In addition, methods employing two or more specific capture probes in sequential captures can be readily devised. The final capture on a nylon filter serves two purposes: It enables autoradiography to be used in place of scintillation counting to quantitate faint but detectable signals. In addition, the extreme concentration of the target into 2 mm’ enables the detection of as few as 700 “P decay events (27). With a 30% overall efficiency of capture of labeled targets and a 770-mer riboprobe containing approximately 35 atoms of “P, about 60 target molecules (lO-22 mol) initially present would be detectable if nonspecific backgrounds are controlled sufficiently well (10.5 logs of background reduction with 1.6 X 10” probes input). The use of solvents that greatly accelerate hybridization (28) can significantly reduce the amount of input probe. This should also be a promising avenue to improve the detection limit with the current background reduction of 7-8 logs. In addition, the use of direct background capture procedures, which is described in the accompanying paper (25), can also improve the background reducing capabilities of RTC that were described in this paper. A nonradioactive method of detection of the captured target would be desirable in order to reduce the total as-

358

MORRISSEY

ET

AL.

say time. However, no nonradioactive detection method that is as sensitive as radioisotopic detection has yet been found for targets captured on either magnetic beads or nylon filters. In the previous paper (14) a nonradioactive method of detection on polystyrene was described that is as sensitive as radioisotopic detection on polystyrene (after one round of capture). In the accompanying paper (25) a nonradioactive method of detection on nitrocellulose is described that is almost as sensitive as radioisotopic detection (after three rounds of capture). APPENDIX

A

The Stability of Poiy(dA) and Poly(rA) as a Function of the Concentration of GuSCN at 37’C In a previous paper (14), it was shown that the binding of poly(rA)-but not poly(dA)-to poly(dT) is quantitatively inhibited in 2.5 M GuSCN at 37OC. It has also been shown that the binding of poly(rA) to oligo(dT) magnetic beads is inhibited by high concentrations of GuSCN at room temperature and 37OC! (29). However, nothing was established about the mechanism. For example, the binding of poly(rA) may be much slower than the binding of poly(dA) or the poly(rA)-oligo(dT) helix may be selectively destabilized in GuSCN. The experiments performed below show that (i) the failure of poly(rA) to bind to oligo(dT) in 1.0 M GuSCN at 37’C is due to the instability of the poly(rA)-oligo(dT) duplex in GuSCN and (ii) they define the concentrations of GuSCN that permit target capture with dA-tailed probes and target elution from the magnetic beads at 37C. Magnetic beads were incubated for 5 min at 37OC!with either 5’-end-labeled poly(rA) or poly(dA) in a buffer containing either 0.5 M NaCl (control) or GuSCN in the range of 0.25 to 1.5 M. The beads were washed twice with the indicated buffer at 37’C and scintillation counted. The results are shown in Fig. 9A. The data of Fig. 9A show that the binding of to oligo(dT) magnetic beads is strongly inwMN4~ hibited (>95%) in GuSCN at 1.0 M or above at 37OC. On the other hand, poly(dA) binds about as well (>95%) to magnetic beads in 1.0 M GuSCN as in 0.5 M NaCl, but gradually loses the ability to bind at higher GuSCN. Thus by capturing the targets on the beads in about 1.0 M GuSCN at 37C, endogenous poly(rA) can be effectively blocked from binding to the oligo(dT) beads. To see if the poly(rA)-oligo(dT) helix is unstable in 1.0 M GuSCN or if the rate of formation of this helix is just unusually slow, poly(rA) was bound to oligo(dT) beads in the 0.5 M NaCl buffer and washed twice with the various GuSCN buffers at 37°C. The results are shown in Fig. 9B. The data of Fig. 9B are essentially indistinguishable from the data of Fig. 9A and thus strongly suggest that the poly(rA)-oligo(dT) helix is simply unstable in 1.0 M GuSCN. Perhaps the poly(rA)

-.lGuSCN1, LlPoly~dAbOligo~dT~

M

l

PolykAkOligoCdT1

FIG. 9.

(A) The binding of poly(rA) and poly(dA) to oligo(dT) magnetic beads as a function of [GuSCN]. P01y(dA)~ (0.1 pmol) was incubated for 5 min in a total volume of 0.1 ml with oligo(dT) beads containing 1.2 pmol of oligo(dT) at 37’C in the presence of various concentrations of GuSCN. The beads were incubated two times for 2 min each with these same buffers at 37’C and the fluid was discarded. The beads were resuspended in 0.6 ml bead wash buffer and scintillation counted. The amount of label bound to the beads in the 0.5 M NaCl control was taken as 100% binding for both polymers and is indicated as the 0 M GuSCN point on the curves. Poly(rA) was similarly performed with 5’-end-labeled poly(rA)4W, except that 0.025 pmol of polymer was added to the beads. (B) The stability of poly(rA) and poly(dA) bound to oligo(dT) beads as a function of the GuSCN concentration in the wash buffer. The indicated polymers were bound to the oligo(dT) beads in 0.5 M NaCl at 37OC and washed twice with the buffers containing the indicated concentration of GuSCN at 37-C. The beads were resuspended in 0.6 ml of bead wash buffer and counted.

can form an intramolecular secondary structure in 1.0 M GuSCN involving hundreds of A-A “base pairs” (30) that is significantly more stable than the short basepaired competing structure, oligo(dT)-oligo(rA). Upon formation of the proposed secondary structure, the oligo(dT) magnetic bead would be displaced by the poly(rA) folding back upon itself. These data also imply that target capture at 37’C with dA-tailed probes will be inhibited at a GuSCN concentration of greater than 1.0 M and that target elution will occur at a GuSCN concentration of 2.0 M or greater. This is indeed the case: Target capture with dA-tailed probes exactly parallels the behavior of dAsO (data not shown). Thus the binding of dAsO to the beads may be used to conveniently study the ability to bind targets to the beads under various conditions. The data of Fig. 9 explain why capture is performed in 0.83 M GuSCN (2 vol of beads is added to 1 vol of sample containing 2.5 M GuSCN) and why chemical elution is performed in 2.5 M GuSCN at 37‘C.

dA-TAILED APPENDIX

CAPTURE

B

Key Formulas Used to Predict Detection Limits of the Assay as a Function of the Efficiency of Hybridization and the Degree of Noise Reduction The signal/noise ratio (S/iv) number (7’) may be written as

as a function

of target

2. 3. 4. 5.

PROBES, Gillespie,

D., and Spiegelman,

S. (1965)

Southern,

E. M. (1975)

Biol.

Ruth,

[2] L+(T) = cpm (T)/Sa = Tiger, where &, is the specific activity of the probe in cpm/molecule, Ti is the initial number of targets, and E,, is the overall efficiency of capture. [3] L-(O) = cpm(contro1 Cl)/Sa unless otherwise specified. [4] L-(O) = Li/lOLoG’B’ = (cpm input/Sa)/lOLoG(B), where Li is the initial number of labeled probes added to the sample, and LOG is the number of logs of background reduction afforded by the procedure. [5] II(k) = Ti(S/~ = /z), where II is the definition of the detection limit as the initial target number (Ti) at which the S/N ratio is equal to k. k = 3 is taken as the cutoff for statistical significance in these papers. [6] I = (k*Li)/(E~~ lOLoG’B’). [7] D(kl) = D(kJ * (kJk& provided E. is constant between T = Ti(l) and Ti(2). For Ti = 120,000 and E. = 0.30 (from a regression analysis of a plot of the average number of ternary complexes captured on the nylon filter vs initial target number), Li = 1.3 X 10” labeled probes input, and LOG(B) = 7.3 (average of two triple capture values of Table 5), k would equal 5.5. With 20,000 ribosomal RNAs per Listeriu, this would imply that six Listeriu cells should be readily detectable at about 5.5 times the background. Compare the data of Fig. 7C, where six Listeriu cells were detected at k = 4 times the background.

Gillespie (Hahnemann University) for his and hybridization in chaotropic salts. We suggesting Biomag as the principal solid We thank Jay Shaw for defining suitable low concentrations of GuSCN.

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We are grateful to David help with sample processing thank Donald Mahan for phase for performing RTC. hybridization conditions in

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ACKNOWLEDGMENTS

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[1] S/N(T) = L+(T)/,%(T = 0) where L+ is the number of labeled probes associated with T (captured) targets, and L- is the number of labeled probes detected in the sample not containing any target molecules (T = 0).

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