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www.nature.com/naturebiotechnology • SEPTEMBER 2002 • VOLUME 20 • nature biotechnology
Beyond gene containment
To the editor:We are writing to you on behalf of the UKstatutory conservation agencies, whichwelcome your editorial “Going with theflow” (Nat. Biotechnol. 20, 527, 2002).
For several years, we have been advocat-ing gene-containment strategies to add tothe environmental safety of novel crops.Note that we refer to “novel” crops and notto genetically modified(GM) crops, as we agreewith your view that this isnot an issue that appliessolely to transgenic crops.However, we believe it isnecessary to consider awider context than geneflow per se to avoid plac-ing too much reliance oncontainment technology.It is entirely possible that,rather than seeing genecontainment as an addedsafety mechanism, somebiotechnologists may seethe technology as a “greenlight” to introduce poten-tially risky genetic traits that otherwisemight be rejected by regulatory authorities.This regulatory problem needs to beresolved.
Perhaps the most obvious and simplegene-containment strategy, often over-looked in these debates, is choosing theright plant for transformation. Thereseems to be a trend toward transformingfood plants (especially corn and oilseeds)for pharmaceutical and industrial feed-stock traits that, even with effective genecontainment, will cause public concernover the adulteration of basic foods. Someof these plants can also outcross to wildancestors. If we want to produce “designer”molecules from agriculture, why notchoose crops that have no sexually compat-ible relatives in the intended market area?By choosing the right plant, the develop-ment of gene-containment mechanismsmay be unnecessary—evolution and plantbreeding have already done the job.
It is also important to consider just howeffective gene-containment strategiescould be. We agree with you that the poten-tial of these technologies should beresearched as thoroughly as possible, butthis must include rigorous and transparentdetermination of their fallibility beforethey are used as mechanisms to containnovel traits. Of the molecular-containmenttechnologies currently being researched(Nat. Biotechnol. 20, 581, 2002), chloro-plast transformation is one of the mostpromising. Although it is highly effective insome model plant species, it is not veryeffective in other plants, including severalcrops exhibiting a degree of paternal inher-itance1. The potential “leakiness” of thistechnology also applies to others, such asmale sterilization.
Assuming that gene-containment strate-gies become a practical option, it may bethat different containment technologieswill need to be tailored to individual crops.
It is likely that at least twostrategies with entirelydifferent mechanismsmay be necessary to pro-vide sufficient contain-ment in any one crop. Inthe United Kingdom, wecall this a “belt andbraces” approach. Ifadopted, it could help toinspire confidence inpoliticians and the public.After all, such anapproach has been adopt-ed by many other indus-tries, such as electricalengineering and cardesign, where backup
safety precautions are standardly installed,even when they are not scientifically justi-fied or required by law.
We see the potential for cross-pollina-tion and gene stacking in crops and/or wildrelatives as a difficult and long-term regu-latory challenge in the commercial releaseof novel crops. When one novel crop getsregulatory approval, it may be followed byfurther release of the same crop possessingdifferent and sometimes multiple noveltraits. We have seen this in the largeincrease in genetic transformations of cornand oilseeds globally. The incidence ofuncontrolled gene stacking and the conse-quent potential impact on agriculture andecology are ill understood because very lit-tle research is being done in this area.Because of lack of data, global regulatorysystems controlling novel crop release fallshort of proper consideration of the envi-ronmental impacts of gene stacking. Giventhe lack of public and private investment in
biosafety research generally (Nat.Biotechnol. 20, 542, 2002), it is difficult tosee how we can properly assess the cumula-tive impact on the environment of hybridplants resulting from the release of differ-ent novel varieties of the same crop, letalone hybrids between and within wildplant–crop complexes.
In light of the continuing controversysurrounding GM crops in the UnitedKingdom and other European countries, aswell as public mistrust of scientists and theagricultural industry, there is every reasonto adopt precautionary techniques that canadd to agricultural sustainability and safetyin developing novel crops. If industry con-tinues to ignore the gene-flow issue, thepublic may eventually turn their backs onthe use of novel gene technology in agricul-ture.
Brian Johnson and Rebecca Dallimore,Biotechnology Advisory Unit
English NatureTaunton, UK
1. Advisory Committee on Releases to theEnvironment: Sub-group on Best Practice in GMCrop Design. Guidance on Principles of BestPractice in the Design of Genetically ModifiedPlants. (Department for the Environment, Food &Rural Affairs, London, 2001).
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Letters may be edited for space and clarity.They should be addressed to:CorrespondenceNature Biotechnology345 Park Avenue SouthNew York, NY 10010-1707, USAor sent by e-mail to [email protected] include your telephone and fax numbers.
QC in antisense oligo synthesis
To the editor:Interest in oligonucleotide antisense thera-peutics has regained momentum1,2. Oneantisense therapeutic, Vitravene, has beenapproved, 12 are in clinical trials1, and oth-ers are in various planning stages3. High-quality chemical synthesis of antisenseoligonucleotides via nucleobase and sugar-protected phosphoramidites is crucial tothe expectations of low toxicity, reducedside effects, and low costs2. However, nei-ther the coupling reaction producing thegrowing polymer chain nor the subsequentdeprotection of the full-length oligonu-cleotide occurs with 100% efficiency4.Thus, quality and regulatory concernsabout antisense therapeutics have beenexpressed by scientists at the Food andDrug Administration (FDA; Rockville,MD)5.
Incomplete deprotection of nucleoside-reactive groups could be responsible for theunexplained results observed in the early invitro and cellular stages of drug discovery2.It could also be responsible for immunolog-ical responses seen at high doses in animalmodels and clinical trials2 and thus con-tribute to erroneous conclusions about
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nature biotechnology • VOLUME 20 • SEPTEMBER 2002 • www.nature.com/naturebiotechnology
CORRESPONDENCE
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drug efficacy. The protecting groups, andnot the nucleic acid itself, can elicit an anti-body response6.
Unfortunately, no simple, reproducible,sensitive, and inexpensive analyticalmethod exists to identify and quantifyevery protecting group that may remain inan antisense sample. High-performanceliquid chromatography (HPLC) nucleosidecomposition analysis identifies and quanti-fies protecting groups remaining onoligonucleotides. However, the analysis isinsensitive because it depends on enzymat-ic cleavage of the oligonucleotide and onUV diode-array detection for identifica-tion and quantification. Capillary elec-trophoresis and mass spectrometry detectthe aborted sequences, but are not easilyadapted to identifying and quantifying theprotecting groups that remain on theoligonucleotide6.
To address this problem, we previouslydeveloped monoclonal antibodies (mAbs)
for the specific identification and quantifi-cation of the nucleobase and sugar protect-ing groups commonly used in DNA andRNA chemical syntheses6. Using thesemAbs, we now present a dot-blot assay anda microplate enzyme-linked immunosor-bent assay (ELISA) for identification andquantification of protecting groups thatremain in standard, intact DNA and RNAoligonucleotide samples (Fig. 1).
The mAbs detect as little as 8 pmol of thespecifically protected nucleoside in intactDNA or RNA composed of 160 nmol of thedeprotected nucleoside. Thus, the mAbanalysis is able to detect a single protectednucleoside in oligonucleotide samples con-taining 2 × 104 deprotected nucleosides. Incontrast, HPLC nucleoside-compositionanalysis of enzyme-hydrolyzed DNA is lim-ited to the detection of 2–5 nmol of pro-tected nucleoside6. Using our present mAbdot-blot assay, 5 of 16 commercial DNAproducts obtained from eight differentcompanies are found to have 1.0–5.2%contamination from benzoyl- and iso-propylphenoxyacetyl-protecting groups(Fig. 2A).
Monoclonal antibodies have the advan-tage of identifying specific protectinggroups that remain on intact oligonu-cleotides independent of the base or sugar.The assays are amenable to robotic analysisof hundreds of samples and should beapplicable to oligonucleotides on solidsupports. The antibodies could be used forthe synthesis of affinity columns to sepa-rate incompletely deprotected nucleic acidfrom completely deprotected molecules.
Because the mAb reagents are group spe-cific and not influenced by the polymersupport, as demonstrated by the identifica-tion of 4,4′-dimethoxytrityl groups oncyclodextrin (Fig. 2B), the technology canbe applied to the detection of protectinggroups remaining from the synthesis of
other biopolymers, dendrimers, andbiopolymers on solid supports, such asoligonucleotide and peptide arrays.
Paul F. Agris,Susanna Smith,
and Chi Fu,Molecular and Structural Biochemistry,
and Stephen G. Simkins,Microbiology, Pathology, and Parasitology,
North Carolina State University,Raleigh,
NC 27695([email protected])
1. Braasch, D.A. & Corey, D.R. Biochemistry 41,4503–4510 (2002).
2. Dove, A. Nat. Biotechnol. 20, 121–124 (2002).3. http://www.ClinicalTrials.gov.4. Gilar, M. Anal. Biochem. 298, 196–206 (2001).5. Kambhampati, R.V., Chiu, Y.Y., Chen, C.W. &
Blumenstein, J.J. Antisense Res. Dev. 3, 405–410(1993).
6. Fu, C., Smith, S., Simkins, S.G. & Agris, P.F. Anal.Biochem. 306, 135–143 (2002).
Figure 1. MAb detection of isobutyl protectionon oligonucleotides. (A) Nitrocellulose dot-blotassay. (B) Microplate-based ELISA. MAbdeveloped against the isobutyl-protectinggroup selectively recognizes a 20-meroligonucleotide comprising isobutyl-protecteddG. Protected homopolymer 20-mer standardsvalidated by nucleoside composition andspectral analyses as having 80–95%protection included: isobutyl-protected dG(lane 1); isopropyl-phenoxyacetyl-protected dG (lane 4); benzoyl-protected dC (lane 5);and 8) 4,4′-dimethoxytrityl-protected dT (lane8). The corresponding 20-mer homopolymerstandards validated by HPLC as deprotectedfrom the same protection groups were thefollowing: dG from isobutyl group (lane 2); dGfrom isopropyl-phenoxyacetyl group (lane 3);dC from benzoyl group (lane 6); and dT fromdimethoxytrityl group (lane 7).
Figure 2. MAb detectionof the benzoyl-protectinggroup on commercialsamples, and thecommon hydroxyl-protecting group,dimethoxytrityl, oncyclodextrin. (A) In a dot-blot assay, the mAb against benzoyl groupdetects the contaminatingprotecting group onsamples of 20-mer primerDNA purchased from twodifferent companies (#2
and #6; B, blank lane). (B) The mAb against dimethoxytrityl group detects the protecting group oncyclodextrin using a dot-blot assay even though the immunological response to generate theantibody was initiated with an oligonucleotide derivative. Lane 1, cyclodextrin; lane 2,dimethoxytrityl-derivatized cyclodextrin. The amount of dimethoxytrityl coupled with cyclodextrinwas determined by comparison of absorbance at 500 nm with that of a standard curve fordimethoxytrityl chloride.
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Erratum
The need for national centers for pro-teomicsRuedi Aebersold and Julian D. WattsNat. Biotechnol. 20, 651 (2002).
Because of a proofreading error, Julian D.Watts’ name was incorrect in the authoraffiliations.
The correct affiliations are as follows:Ruedi Aebersold is cofounder and JulianD. Watts is a senior research scientist,Institute for Systems Biology, 1441North 34th Street, Seattle, WA 98103([email protected]). Weregret the error.
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