THE JOTJRNAI. OF BIOLOGICAL CHEMISTRY Vol. 256. No. 2, Issue of January 25, pp. 1023-1028,1981 Prrnted in U S A .

A General Method for Detection and Characterization of an mRNA using an Oligonucleotide Robe*

(Received for publication, August 29, 1980, and in revised form, October 1, 1980)

Kan L. AgarwalS, Janne Brunstedt, and Barbara E. Noyes From the Department of Biochemistry a n d the Department of Biophysics and Theoretical Biology, The University of Chicago, Chicago, Illinois 60637

A general method for the detection and characteri- zation of an mRNA using an oligodeoxynucleotide probe is described. The results presented indicate that a G-dT or a dG-U base pair within a short DNA-RNA hybrid does not significantly reduce the stability of the hybrid. On this basis, we propose that 11 amino acids, including Trp and Met, can be used in selecting a pep- tide sequence suitable for use in designing an oligo- deoxynucleotide probe complementary to a given mRNA. To test this hypothesis, we have synthesized an oligodeoxynucleotide (oligo 11) complementary to the region of gastrin mRNA coding for Trp-Met-Asp-Phe and have compared its effectiveness as a hybridization probe and as a primer for the synthesis of gastrin- specific cDNA with another oligonucleotide (oligo I) complementary to the region of gastrin mRNA coding for Trp-Met-Glu-Glu. Unlike oligo I, oligo I1 functions as a primer in specific cDNA synthesis only when the mRNA is denatured prior to initiation of synthesis. Similarly, oligo I1 can be used as a specific hybridiza- tion probe for gastrin mRNA only when the RNA is denatured and partially cleaved with NaOH before hy- bridization. A simple procedure for denaturing gastrin mRNA to ensure efficient interaction with oligodeox- ynucleotide probes is described. This procedure should be useful in studies with other oligonucleotides and mRNAs as well.

Analysis of transcriptional and translational control mech- anisms in differentiated eukaryotic cells has been hampered by the inability to isolate many mRNAs of known function, particularly those mRNAs present in minute quantities. Con- ventional procedures available for the detection, isolation and characterization of cytoplasmic mRNAs have involved the use of cell-free translation systems in conjunction with immuno- radiometric assays to identify specific protein products. This approach may not be suitable, however, for study of mRNA species directing the synthesis of proteins which undergo extensive post-translational modification. In such cases, the available antibodies are directed against the processed protein and may not interact efficiently with a primary translation product synthesized in uitro. Furthermore, many proteins of interest are available in such small quantities that specific antibodies cannot be readily obtained. To overcome these difficulties, we have developed an alternative approach for

* This work was supported by National Institutes of Health Grants GM 22199 and AM 21901. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

# United States Public Health Service Research Career Develop- ment Awardee (GM 224). To whom correspondence should be ad- dressed.

studying mRNAs which constitute a small proportion of the total cell mRNA (1). The principle of the approach involves the use of a complementary oligodeoxynucleotide probe whose nucleotide sequence can be deduced from the known amino acid sequence of a given peptide. In our initial studies with gastrin, we selected the sequence Trp-Met-Glu-Glu as a suit- able sequence to use for the deduction of a nucleotide probe sequence. Since both Trp and Met have only one codon, their selection minimizes possible ambiguities in the oligodeoxy- nucleotide probe sequence. There are two codons for glutamic acid, however, GAA and GAG, and we selected the statistically favored GAG codon in designing the gastrin mRNA probe. The synthetic oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C- A), oligo I, did serve as a specific probe for the detection and characterization of gastrin mRNA (1) and suggested that the approach could be useful for studying other mRNAs if amino acids other than Trp and Met could be used for the deduction of an oligonucleotide probe sequence.

In this communication, we present data to support the proposition that any of 11 amino acids, including Trp and Met, present in a tetrapeptide sequence in a given protein can be used to deduce an oligodeoxynucleotide probe sequence. To test this hypothesis and to investigate other factors essen- tial for efficient interaction of short oligodeoxynucleotides with RNAs, we synthesized an oligodeoxynucleotide d(G-A- A-G-T-C-C-A-T-C-C-A), oligo 11, which is complementary to a different region of the gastrin mRNA. This oligonucleotide, complementary to the mRNA coding for the tetrapeptide Trp-Met-Asp-Phe, was designed to maximize the stability of the oligo. RNA complex even though the correct codons for Asp and Phe in this sequence are not known. In addition, we present procedures for reducing the interference of mRNA secondary structure in interactions between the mRNA and the oligonucleotide probe, thus increasing the efficiency of cDNA synthesis when oligonucleotides are used as primers and the efficiency of hybridization when oligonucleotides are used as probes.

EXPERIMENTAL PROCEDURES

Enzymes and Reagents-Reverse transcriptase (RNA-dependent DNA polymerase) from avian myeloblastosis virus was provided by Dr. Beard (Life Sciences, Inc., St. Petersburg, FL), and exonuclease- free T4-polynucleotide kinase (32,000 units/mg) was isolated by a modified procedure of Panet et al. (2). Calf intestine alkaline phos- phatase was from Boehringer Mannheim. TI-ribonuclease was from Sankyo. Oligo(dT)-cellulose was prepared as described by Gilham t‘t al. (3). 1-[(m-Nitrobenzy1oxy)methyllpyridinium chloride was pur- chased from Pierce Chemical Co. and used in the preparation of

imide (DCC) was purchased from Aldrich Chemical Co. [y-’”P]ATP DBM-paper as described by Alwine et al. (4). Dicyclohexylcarbodi-

(specific activity 1300 to 1500 Ci/mmol; 1 Ci = 3.7 X 10“’ becquerels) was synthesized as described by Maxam and Gilbert (5).

Homo-oligonucleotides-Oligodeoxycytidylic acid ( d C d , oligode- oxythymidylic acid ( d T d , and oligodeoxyadenylic acid (dA,,,) were

1023

1024 mRNA Detection and Characterization

obtained from P.-L. Biochemicals and oligodeoxyuridylic acid (dug) was from Collaborative Research.

Oligo-5-bromodeoxyuridylic acid (9 to 10 nucleotides in length) was chemically synthesized by polymerization of 5-bromodeoxyuridine 5‘- phosphate in the presence of dicyclohexylcarbodiimide and the reac- tion products were separated by DEAE-cellulose chromatography as described by Khorana and Vizsolyi for the synthesis of oligodeoxy- thymidylic acid (6). Fractions eluting between 0.32 to 0.36 M NaCl were analyzed for oligomer size. A portion of each fraction was desalted, treated with calf intestine phosphatase to remove 5”phos- phates, and 5”labeled with T,-polynycleotide kinase in the presence of [y-,”2P]ATP (7). Each sample was analyzed by electrophoresis on a 20% polyacrylamide, 7 M urea gel, and the fractions with the mobility of bromphenol blue were pooled and used in the preparation of oligonucleotide-cellulose.

Oligoriboguanylate (9 to 11 nucleotides in length) was prepared by partial cleavage of polyriboguanylate with TI-ribonuclease (8). The oligomers thus obtained were 5’-labeled with T4-polynucleotide kinase and [y-’”PIATP. The mixture of labeled polymers was purified by chromatography on Sephadex G-50 (superfine) in 20 mM triethylam- monium bicarbonate. Excluded labeled material was recovered and lyophilized. Further size purification of oligoribogua.1ylate was accom- plished by electrophoresis on a 20% polyacrylamid, gel (40 X 20 X 0.15 cm) in 7 M urea. The radioactive material with the mobility of bromphenol blue was isolated by elution as described by Maxam and Gilbert (5). The oligoriboguanylate thus isolated had a specific activ- ity of 2.2 X io4 cpm/pg.

The oligodeoxynucleotides, d(C-T-C-C-T-6-C-A-T-C-C-A), oligo I, and d(G-A-A-G-T-C-C-A-T-C-C-A), oligo IE, were chemically synthe- sized. Oligo I was synthesized by the diester approach (9) and oligo 11 was synthesized by the triester approach (IO). The fully deblocked oligonucleotides were purified to near-homogeneity by ion exchange chromatography followed by reverse phase chromatography (II), and the nucleotide sequences determined by two-dimensional homochro- matography (12) are in complete agreement with the assigned struc- tures.

Preparation of Oligodeoxynucleotide-Cellulose-The 5‘-phos- phate-terminated oligodeoxynucleotides, pdC,,,, pdTlu, pdU9, pBrdU9, and pdAll,, were linked covalently to cellulose paper using the water- soluble carbodiimide procedure described by Astell et al. (13). Ap- proximately 10 to 15 optical density units (260 nm) of each nucleotide were incorporated per g of Whatman No. 3MM paper.

Column Chromatography of Oligoriboguanylate on Several Oli- godeoxynucleotide-Celluloses-Each oligonucleotide-cellulose paper strip (1 .1 g) was pulverized, packed under pressure into a jacketed column (6 X 18 mm), and equilibrated with 100 mM KHzPO, (pH 7.0) buffer containing 1 mM EDTA and the desired amount of NaCI. 5‘- Labeled oligoriboguanylate (10 pg, 2.2 X IO5 cpm) was adsorbed to each column at 15°C in the presence of 100 mM or 500 mM NaCI. Elution of oligoriboguanylate was accomplished with the same buffer using a linear temperature gradient giving an increase of 0.8”C/10 min/0.75-ml fraction as previously described (13). Radioactivity in each fraction was determined by Cerenkov counting.

Preparation of Poly(A)-RNA-Antral mucosa from freshly slaugh- tered hogs was dissected from the pyloric gland area and immediately frozen in liquid nitrogen. Total nucleic acids were isolated from the tissue as described previously, and RNA free of DNA and proteins was recovered following centrifugation through CsC12 (1). A poly(A)- enriched RNA fraction was isolated by repeated chromatography on oligo(dT)-cellulose (14).

5”Labeled Oligonucleotides-The oligonucleotides (1 nmol) were 5’-:”P-labeled by using TI-polynucleotide kinase and [y-”’PIATP (7). The labeled oligonucleotides were isolated free of ATP and inorganic phosphate by chromatography on a 10-ml column of Sephadex G-50 (superfine) in the presence of 10 mM ammonium bicarbonate. Frac- tions containing labeled oligonucleotides were pooled and lyophilized several times (three to four times) to remove most of the ammonium bicarbonate. Oligonucleotides to be used as primers for reverse tran- scription were resuspended in 10 m~ sodium phosphate, pH 7.2, 10 mM EDTA, and stored a t -20°C.

cDNA Synthesis-Equal volumes of hog antral poly(A)-RNA in H,O and 5’-labeled oligonucleotide primer (oligo I or oligo 11) were mixed such that the RNA concentration was 100 to 120 pg/ml and the primer concentration was 15 to 20 pg/ml in 5 mM sodium phos- phate, pH 7.2,5 mM EDTA. Samples were treated in one of two ways: 1) The RNA.oligonucleotide mixture was incubated at room temper- ature (25°C) for 30 min and then 1.2 M NaCl was added to raise the salt concentration to 94 mM. The samples were transferred to 4°C

and incubated an additional 30 min. 2) The RNA.oligonucleotide mixture was heated in a capped, 1.5-ml polypropylene tube at 90°C for 5 min (25 pl volumes), or 15 min for 100 pl volumes, and then 1.2 M NaCI, preheated to 90”C, was added to a final concentration of 94 mM. The reaction tube was transferred to a small volume of 90°C water and allowed to cool to room temperature over a period of 15 to 20 min. Samples were then transferred to 4°C. After one of these annealing procedures, the reaction mixture was adjusted to 50 mM Tris-HC1, pH 8.3,5 m~ dithiothreitol, 15 mM MgCle, 100 pg/ml bovine serum albumin, and 500 p~ each of the four unlabeled deoxynucleo- side triphosphates. The final NaCl concentration was 60 mM. cDNA synthesis was initiated a t 4°C with the addition of reverse transcrip- tase (Lot G-180, 12 units/pl) to 480 units/ml. After incubation for 90 min a t 38”C, the reaction was terminated by addition of EDTA to 20 mM followed by phenol/CHCl:$ (l:l, v/v) extraction. The aqueous phase was adjusted to 0.3 M sodium acetate and the nucleic acids were precipitated by addition of 2% volumes of 95% ethanol. The precipi- tate was rinsed two times with 1 ml of 75% ethanol, dried under vacuum for 2 min, dissolved in 0.1 M NaOH, 2 mM EDTA (2 pl/pg of poly(A)-RNA) and incubated a t 37OC for 30 min. An equal volume of 10 M urea, 0.03% xylene cyanol, 0.03% bromphenol blue was added and the mixture was heated a t 90°C for 30 s followed by quick cooling in ice H20.

cDNA samples were analyzed by electrophoresis on an 8% poly- acrylamide, 7 M urea slab gel (40 X 20 X 0.05 cm) prepared as described by Sanger and Coulson (15). Preparative separation of the cDNA products was carried out on a 16% polyacrylamide, 7 M urea slab gel (40 X 20 X 0.15 cm). The labeled cDNA products were visualized by autoradiography as described previously (1).

DNA Sequence Analysis-Labeled cDNA was eluted from the gel and subjected to nucleotide sequence analysis using the chemical cleavage procedure described previously (5). Approximately 40,000 cpm (Cerenkov) were used for the four base-specific cleavages and the products were separated by electrophoresis on an 8% polyacryl- amide, 7 M urea slab gel (40 X 20 X 0.03 cm). Autoradiography of the gel was a t -70°C for 2 days using Kodak No-Screen film.

DNA-RNA Hybridizations-Poly(A)-RNA (5 pg) was electropho- resed on a 1.58 agarose gel in the presence of 4 mM CH:3HgOH. The RNA in the gel was either treated with 100 mM NaOH for 1 h before transfer to DBM-paper, or transferred to DBM-paper directly without NaOH treatment as described by Alwine et al. (4). Hybridization of RNA with [”’PIcDNA was as described previously (1). [“2P]Dodeca- nucleotides (oligo I and oligo 11) were hybridized to RNA as described by Mevarech et al. (16), except that 200 pg/ml of poly(A) was used as carrier.

RESULTS

Column Chromatography of 5’4abeled Oligoribogua- nylate on Oligodeoxynucleotide-Cellulose-The previous ob- servation of Gillam et al. (17) that G-dT base pairs in short DNA-RNA hybrids do not significantly alter hybrid stability, induced us to investigate the stability of other RNA-DNA base pairs which might occur in interactions of synthetic oligodeoxynucleotides with mRNA. Specifically, we have studied the thermal elution of oligoriboguanylate (G9-11) from several different oligodeoxynucleotide-cellulose columns at two salt concentrations, 100 m~ and 500 m~ NaCl. These salt concentrations were selected because they are similar to those often used in oligonucleotide-primed cDNA synthesis and oligonucleotide hybridization with mRNA. Several oligodeox- ynucleotides, pdAlo, pdTlo, pBrdU9, and pdClo, were cova- lently coupled to cellulose, and 1-ml columns of each were prepared. 5’-Labeled G9.11 was passed through each column at 15OC. All of the radioactive sample applied passed through the pdAlo column, but in each of the other cases all of the G9_ll was retained. This material was eluted with a linear temperature gradient, and the elution temperature for each was determined from the midpoint of the radioactive peak profiie. The results, given in Table I, indicate that oligoribo- guanylate interacts with oligodeoxypyrimidines, but does not interact significantly with oligodeoxyadenylate under the ex- perimental conditions used. This suggests that purine-purine interactions in RNA-DNA hybrids are not favorable. How-

mRNA Detection and Characterization 1025

TABLE I Thermal elution of oligoriboguanylate (G9-11) from

oligodeoxynucleotide-cellulose columns Ribooligonucleo- Oligodeoxynucleotide- NaCl ( 1 0 0 mM) NaCl ( 5 0 0 mM)

tide cellulose elution temp. elution temp.

112 pG9.11 Cellulose-pdCt0 65 nd" :VZ pG,.ll Cellulose-pBrdU9 52 72 :12 pG9-ll Cellulose-pdU9 48 58

:12 pG9-ll Cellulose-pdAl0 na nah :'2pGs-l, Cellulose-pTIII 42 51

" nd, not determined. na, not adsorbed.

ever, the guanine residue does interact with pyrimidines, and the stability of the interaction varies with different deoxypy- rimidine residues. For instance, Ge-l I interacts specifically with dTln in 100 mM and 500 mM NaCl requiring temperatures of 42°C and 51"C, respectively, for elution. The stability of interaction is increased appreciably, however, when the deox- ynucleoside residue is dU or BrdU. Interaction of G with BrdU in the presence of 500 mM NaCl is as effective as interaction with dC in 100 m~ NaCl. The results clearly show that G interacts with d T (in agreement with the results of Gillam et al. (17)), and that the stability of the interaction with other pyrimidines is in the order dC > BrdU > dU > dT.

cDNA Synthesis and Characterization-5":'2P-Labeled oligo I and oligo I1 (specific activity 2.3 to 2.4 X 10" cpm/ pmol) were prepared by phosphorylation with [y-'"PIATP and T,-polynucleotide kinase and were used in the synthesis of gastrin-specific cDNAs from hog antral poly(A)-RNA. The reaction products were analyzed by electrophoresis on an 8% polyacrylamide gel in the presence of 7 M urea. As shown in Fig. 1, Lanes 3 and 5, only two major cDNA products of large size are obtained when oligo I is used as a primer for reverse transcription of hog antral poly(A)-RNA. The synthesis pat- tern is the same whether or not the RNA and primer are heated and annealed prior to cDNA synthesis. This result is in agreement with our previous findings in which we demon- strated by nucleotide sequence analysis that cDNA-A (304 nucleotides long) is gastrin-specific and cDNA-B (200 nucleo- tides long) apparently is not (1). Although 8 of the 12 nucleo- tides of oligo I are the same as those at the 3"end of oligo 11, we have not detected significant priming by oligo I at the position of oligo I1 or vice versa.

It is important to note that, unlike the results obtained with oligo I, no specific cDNA synthesis can be obtained using oligo I1 as primer unless the RNA is f i t heated at 90°C in the presence of EDTA and primer and allowed to cool slowly (see Fig. 1, Lanes 1 and 2). We have investigated several conditions for cDNA synthesis using oligo I1 as primer, including various concentrations of reverse transcriptase and deoxynucleoside triphosphates and numerous procedures for denaturing RNA and annealing RNA and primer. However, the best results have been consistently obtained when RNA is denatured in the presence of EDTA and primer by heating a t 90°C for 5 min. Omission of EDTA during the heating step results in RNA degradation and thus no cDNA synthesis as shown in Fig. 1, Lane 4. The results indicate that the procedure de- scribed under "Experimental Procedures" for heating the RNA a t 90°C in the presence of EDTA and primer followed by addition of salt and slow cooling gives satisfactory cDNA synthesis with both oligo I and oligo I1 and should be suitable for use with other RNAs and primers.

Based on the amino acid sequence of gastrin (see Fig. 2), gastrin-specific cDNA synthesized using oligo I1 as a primer should be 30 nucleotides longer than the cDNA product obtained using oligo I as a primer for reverse transcription.

766 - 543- 525'

C-

237 - B"

109 -

a3 - FIG. 1. Electrophoretic analysis of cDNA products synthe-

sized from hog poly(A)-RNA using oligo I and oligo I1 primers. cDNAs synthesized from 2.0 pg of poly(A)-RNA using "2P-labeled oligo I (80 pmol, 2.4 X 10" cpm/pmol) and oligo I1 (80 pmol, 2.3 X IO" cpm/pmol) as primers were electrophoresed on an 8% polyacrylamide, 7 M urea gel. Lane I, RNA + oligo I1 + EDTA + 90°C heat treatment; Lane 2, RNA + oligo I1 + EDTA (no 90" heat treatment); Lane 3, RNA + oligo I + EDTA + 90°C heat treatment; Lane 4, RNA + oligo I + 90°C heat treatment in the absence of EDTA, Lane 5, RNA + oligo I + EDTA (no 90°C heat treatment). Nucleotide lengths marked on the left were determined from end-labeled Hinff fragments of SV40 DNA electrophoresed on the same gel.

Fig. 1, Lane I , shows that a prominent cDNA product (cDNA- C) about 337 nucleotides long is obtained when oligo I1 is used to prime cDNA synthesis. To obtain homogeneous cDNA-C suitable for hydridization experiments and nucleotide se- quence analysis, the cDNA synthesis was repeated using 12 pg of RNA, and the products were separated on a 16% poly- acrylamide gel in the presence of 7 M urea. The 337 nucleotide cDNA-C was extracted from the gel and used in hybridization experiments with hog antral RNA and for nucleotide sequence analysis. As predicted, cDNA-C hybridized to the same size RNA (620 nucleotides long) as the gastrin-specific cDNA-A (data not shown). Furthermore, the results of nucleotide se- quence analysis shown in Fig. 2 clearly demonstrate that cDNA-C is also gastrin-specific and that oligo I1 primes the synthesis of cDNA at the expected position on the gastrin mRNA. The data also indicate that the codon used for glu-

1026 mRNA Detection and Characterization 1 5 10 15

Protein (NH?).. . ... Gln - Gly - Pro - Trp - Met - Glu - Glu - Glu - Glu - Glu - Ala - T y r - 61y - Trp - Met - Asp - Phe - NH2 mRNA(5'-) ....,. C A 6 G G G C C A U G G A U 6 G A 6 G A 6 G A A 6 A A 6 A A 6 C G U A U G 6 X U 6 G A U G G A C U U C

cDM-A (3") ...... 6 T C C C C 6 G T A C C T A C C T C C T C - 32P

cDNA-C(3'-) ........................ A C C T A C C T C C T C C T T C T T C T T C 6 C A T A C C X A C C T A C C T 6 A A 6 - 3 2 P

OL160 I

OLIGO I 1 FIG. 2. Partial nucleotide sequence of the mRNA coding for

hog gastrin. The lower line shows the nucleotide sequence of cDNA- C (see Fig 1) derived from hog antral poly(A)-RNA using '"P-oligo I1 as a primer for reverse transcription. The nucleotide sequence previ- ously determined (1) for cDNA-A using oligo I as a primer for reverse

iP & -1765

- 217

FIG. 3. Hybridization of 5'-"P-oligodeoxynucleotides to poly(A)-RNA from hog antral mucosa. I'oly(A)-RNA (5 pg/lane) was electrophoresed on a 1.58 agarose gel in the presence of 4 mM CH,,HgOH. The RNA was transferred to DBM-paper and hybridized to '"€"labeled oligodeoxynucleotides. Lanes A and B show the results of hybridization at 23°C of oligo I (1.2 X lo" cpm; 3.1 X IO" cpm/ pmol) and oligo I1 (9.4 X lo4 cpm; 3.1 X IO" cpm/pmol) with RNA which was hydrolyzed with 100 mM NaOH before being transferred to DBM-paper (4). Lane C shows the results of hybridization at 23°C of oligo I1 (9.4 X IO4 cpm; 3.1 X 10" cpm/pmol) with RNA transferred to the paper without prior base hydrolysis. 5'-Labeled Hind111 frag- ments of SV40 DNA were used as nucleotide length markers and their positions are marked in Lane D.

tamic acids 6 and 7 is GAG, and therefore, oligo I is comple- mentary to gastrin mRNA with no mismatched base pairs.

Hybridization of Oligonucleotides with Gastrin mRNA- 5'-:'"P-Labeled oligo I and oligo I1 were hybridized to hog antral poly(A)-RNA which had been electrophoretically sep- arated on a 1.5% agarose gel in the presence of 4 mM CHRHgOH and transferred to DBM-paper (4) . Previously, we showed that oligo I hybridizes efficiently to a mRNA about 620 nucleotides in length and that no difference in the ef i - ciency of hybridization could be detected between RNA sam- ples which were partially hydrolyzed with base prior to trans- fer from the gel and those that were not (16). The data presented in Fig. 3 clearly show, however, that hybridization of oligo I1 with gastrin mRNA is dependent upon partial base hydrolysis of the RNA prior to transfer. Fig. 3, Lanes A and B, show that both oligo I and oligo I1 hybridize specifically to the same size class RNA when the RNA is treated with 100

transcription is given above the sequence derived from oligo 11. The partial sequence for gastrin mRNA shown below the amino acid sequence, is derived from the two cDNA sequences. The X following the 3'-end of oligo I1 represents a nucleotide which could not be identified with certainty.

mM NaOH for 60 min a t room temperature before transfer. However, as shown in Lane C, no hybridization of oligo I1 with RNA was detected when the RNA was transferred to the DBM-paper without prior base hydrolysis. These results suggest that hybridization of oligonucleotides to partially hy- drolyzed RNA preparations remains specific, and that second- ary structure in certain regions of an RNA molecule may interfere with efficient hybridization of oligonucleotides.

DISCUSSION

Recently, we showed that an oligodeoxynucleotide can be used as an effective probe in the detection of minute quantities of gastrin mRNA (1, 16). Furthermore, oligonucleotide-gen- erated cDNA can be used as a specific hybridization probe in the selection of recombinant DNA molecules containing a specific cDNA sequence (18). These studies showed that oligonucleotides as short as a decanucleotide can serve as a specific primer for the s-mthesis of cDNA and as a specific hybridization probe. This approach is highly sensitive and allows the detection of as little as 0.2 fmol of gastrin mRNA/ pg of poly(A)-RNA (16). Although this method should be of general application for the detection and characterization of mRNAs corresponding to proteins of known amino acid se- quence, it is limited by the fact that most amino acids are coded for by two or more codons. Thus, one cannot always deduce the mRNA sequence of a short peptide with certainty, or determine the correct complementary sequence desired for a suitable DNA probe. Earlier work (17, 19), as well as the results presented here, indicate, however, that G-dT and dG- U base pairs should not significantly alter the stability of short RNA-DNA hybrids. On this basis we suggest that the ap- proach can be of general use and that any of 11 amino acids including Trp and Met can be used to design a suitable oligodeoxynucleotide probe. These 11 amino acids are given in Table 11. Trp and Met are the most desirable amino acids to include in a chosen sequence since each has a single codon. Nine other amino acids have only two codons, the ambiguity being in the third position. For six of these amino acids, the third nucleotide in the codon is either U or C. If one always selects dG for the complementary probe sequence, the probe will carry either a stable dG-C base pair or a dG-U base pair, which is less stable but which should not significantly alter the stability of interaction of the oligodeoxynucleotide with the RNA. Similarly, for the three amino acids with either an A or G in the third position of the codon, one should always select d T or an analog of d T for the complementary probe sequence. This ensures that the probe carries an accepted dT- A base pair or an allowed, although less stable, dT-G base pair.

To test this hypothesis, we have begun to examine the ability of various oligodeoxynucleotides to function as specific probes for gastrin mRNA. In this communication, we have shown that two dodecanucleotides complementary to different

mRNA Detection and Characterization 102 7

TABLE I1 Amino acids useful in the deduction of an oligodeoxynucleotide

probe sequence Amino Deduced Amino

acid RNA DNA codon acid RNA codon DNA codon Deduced

Phe ::: AAG ‘ln CAG

ATG Lys AAA AAG TTT

GTG Glu GAG GAA CTT

CAA GTT

Tyr UAC

His CAC

AAC AAu TTG

Asp GAC GAU CTG Trp UGG ACC

Cys ::: ACG Met AUG TAC

regions of gastrin mRNA (see Fig. 2) can be used as hybridi- zation probes and as primers for the synthesis of gastrin- specific cDNA. Oligo I, d(C-T-C-C-T-C-C-A-T-C-C-A), is complementary to the region of gastrin mRNA coding for Trp-Met-Glu-Glu and is complementary to the mRNA with no mismatched base pairs. Oligo 11, d(G-A-A-G-T-C-C-A-T- C-C-A), is complementary to the region of gastrin mRNA coding for Trp-Met-Asp-Phe and could contain one or two mismatched base pairs. That is, in choosing the sequence for oligo 11, we followed the proposal presented above and selected dG as the residue complementary to the third position nu- cleotide for the codons for Asp and Phe (see Table 11). Both oligo I and oligo I1 can be used successfully to detect gastrin mRNA within a heterogeneous pool of RNAs and to synthe- size gastrin-specific cDNA. However, the efficiency of gastrin- specific cDNA synthesis is about 2-fold higher when oligo I is used as a primer relative to that obtained with oligo 11. This difference may be due in part to a lower affinity of oligo I1 for gastrin mRNA because of one or two dG-U base pairs in the hybrid. However, our experiments suggest that a more likely explanation is very stable secondary structure in the gastrin mRNA which limits access of oligo I1 to the hybridization site. For example, oligo I efficiently primes the synthesis of gastrin cDNA whether or not the RNA is denatured by heating and annealed with primer. Oligo 11, on the other hand, does not prime specific cDNA synthesis unless the RNA is denatured and pre-annealed with oligo I1 (see Fig. 1). Furthermore, the efficiency of synthesis of cDNA-C changes with changes in the RNA denaturation procedure. We have tried denaturing the mRNA preparation with formamide, CHsHgOH, and heat- ing in the presence of EDTA, all followed by either slow annealing with primer or rapid chilling and addition of primer. The best results have consistently been obtained with the procedure outlined under “Experimental Procedures” in which RNA is heated a t 90°C in 5 mM EDTA and allowed to cool gradually in the presence of primer and salt. The presence of EDTA is essential during heat denaturation of RNA for satisfactory results. In addition, oligo I hybridizes to intact RNA bound to DBM-paper while oligo I1 will not hybridize under the same conditions unless RNA is partially hydrolyzed with NaOH prior to transfer to the diazotized cellulose paper. All of these experiments indicate that secondary structure in mRNAs is a critical parameter to consider in designing exper- iments with oligodeoxynucleotide probes. A synthetic probe with the correct sequence may not function as an efficient probe for a given RNA if the complementary sequence of the

RNA is involved in relatively stable intrastrand interactions. Even after denaturation of the RNA a short oligodeoxynu- cleotide may not be able to compete effectively for the hy- bridization site.

Our results indicate that the extent of stable interaction between an oligodeoxynucleotide and a given RNA will de- pend on several factors, including the nature of RNA second- ary structures, the length of the oligonucleotide probe, the percentage of G-C base pairs in the RNA.oligonuc1eotide complex, and the position of any dG-U base pairs within the template-primer complex. Previously, we showed that an octanucleotide with a 50% G-C content hybridizes less effi- ciently than a dodecanucleotide with a 58% G-C content (16) or a decanucleotide with a 70% G-C content (la).’ On this basis, we suggest that oligonucleotide probes be designed so that they are at least 10 nucleotides long with a G-C content of at least 50 to 55%. Another important factor to consider when choosing an oligodeoxynucleotide probe sequence is the location of any dG-U base pairs in the DNA-RNA hybrid. Although we have not yet specifically investigated this param- eter in detail, our previous work with rat insulin mRNAs showed that a decanucleotide complementary to rat insulin I mRNA functions effectively as a primer for the synthesis of insulin I-specific cDNA, but does not prime synthesis of insulin I1 cDNA (18). Examination of the insulin I1 mRNA sequence shows that if a decanucleotide.RNA complex is formed, there is a dG-U base pair at the 3’-end of the oligo- nucleotide and a dC-A base pair near the 5’-end of the oligo- nucleotide. Although this hybrid would contain seven unin- terrupted, correctly matched base pairs and a G-C content of 57%, we assume that the 3”terminal dG-U base pair is not suitable to allow initiation of cDNA synthesis by reverse transcriptase. Because rat insulin I and I1 mRNAs have not been separated, we do not know whether the decanucleotide can function as a hybridization probe for both mRNA or for only rat I mRNAs. The results suggest, however, that al- though dG-U base pairs may be allowed near the 5’-end of a deoxyoligonucleotide probe, they are not desirable at the 3’- end of an oligonucleotide to be used as a primer for cDNA synthesis.

In this communication, we have attempted to identify some of the many factors involved in the formation of stable oligo- deoxynucleotide - mRNA complexes. Many parameters remain to be more thoroughly investigated, and our efforts continue to improve this potentially useful approach for studying nu- cleic acids which code for peptides of known amino acid sequence.

Acknowledgments-We are indebted to Richard Ferguson for pro- viding hog antral mRNA and to Dr. Moshe Mevarech for many helpful discussions.

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