of the DNA, is more difficult, and for them the error rate in the present study varied between 5% and 30% depending on the frequency of the variant.

Given the declining cost of DNA sequen-cing, future discoveries in human genomics are more likely to be based on a combination of exon sequencing and low-coverage, whole-genome sequencing, rather than on the more traditional techniques. Such DNA sequencing gives access to rare and novel variants, as well as being more suitable for identifying DNA insertions and deletions and, in general, for detecting less-common variants that affect only a single nucleotide.

The remaining question is how to accom-modate errors in low-coverage sequenc-ing, because an error rate of even a few per cent can lead to drastically reduced power if not accounted for appropriately9. Statistical methods that incorporate high error rates will be an essential component of future

genomic-sequencing efforts. But no matter which protocol is used, the focus of the third phase of human genomics will clearly be on whole-genome sequencing. ■

Rasmus Nielsen is in the Departments of Integrative Biology and of Statistics, University of California, Berkeley, Berkeley, California 94720, USA. e-mail: rasmus_nielsen@berkeley.edu

1. www.1000genomes.org/page.php?page=about2. The 1000 Genomes Project Consortium Nature

467, 1061–1073 (2010).3. International Human Genome Sequencing

Consortium Nature 409, 860–921 (2001).4. Venter, J. C. et al. Science 291, 1304–1351 (2001).5. Hindorff, L. A. et al. Proc. Natl Acad. Sci. USA 106,

9362–9367 (2009).6. Hardy, J. & Singleton, A. N. Engl. J. Med. 360,

1759–1768 (2009).7. Manolio, T. A. et al. Nature 461, 747–753 (2009).8. Servin, B. & Stephens, M. PLoS Genet. 3, e114

(2007). 9. Huang, L. et al. Am. J. Hum. Genet. 85, 692–698

(2009).

developed by Chilkoti and colleagues2,8, is to grow the polymer chain on the protein drug by polymerization; this would, in principle, allow any length of polymer chain to be grafted.

Chilkoti and colleagues’ strategy2 has many advantages. To solve the problem of multiple sites of polymer grafting, they used a protein-engineering trick to place a single chemical group at the carboxy terminus of the protein. They used this group to attach an initiator molecule for a polymerization reaction, select-ing a strategically advantageous initiator for a polymerization reaction that gives precise con-trol of polymer length under mild conditions, consistent with the delicate nature of proteins. This allowed the growth of a very long polymer chain, one that looks like a bottlebrush, con-sisting of a long main chain covered by short PEG chains along its length, with the polymer attached to the protein’s carboxy-terminal site.

The result of this convergence of protein and polymer engineering was anything but subtle: the bottlebrush polymer on the car-boxy terminus of the model protein increased its hydrodynamic radius almost sevenfold, from 3 to 20 nanometres — an increase in size corresponding to an almost 300-fold increase in hydro dynamic volume. One can imagine the result as being rather like an elfin dancer adorned with an outrageously long and fluffy feather boa (Fig. 1), rather than a few peacock feathers, as would be the effect using previ-ous approaches. The grafted polymer chain resulted in a substantial prolongation in cir-culation time, which the authors showed to be beneficial in targeting tumours.

D R U G D E V E L O P M E N T

Longer-lived proteinsShort residence times in the bloodstream reduce the effectiveness of protein drugs. Application of an approach that combines protein and polymer engineering prolongs circulation time and increases drug uptake by tumours.

J E f f R E y A . H U b b E L L

The past 25 years have seen an explo-sion in the number of approved protein drugs produced by genetic engineer-

ing, for treating hormonal, metabolic, immu-nological, haematological and reproductive disorders, as well as cancer1. Scientists initially sought to perfectly copy nature’s structural expression of these proteins, leading to many first-generation drugs. Subsequently, protein engineers began to adapt nature’s structures, either subtly (for example, by changing a few amino-acid residues to make interactions with a target molecule stronger or more specific) or more profoundly (for instance, by attaching two unrelated proteins to create a protein pos-sessing a combined function that nature never considered). Several second-generation drugs have resulted from such efforts.

One drawback of protein drugs is their rapid clearance from the systemic circulation. Writ-ing in Proceedings of the National Academy of Sciences, Chilkoti and colleagues2 now describe a combined protein- and polymer-engineering approach to prolong protein circulation and enhance drug accumulation in tumours.

The concept of polymer attachment to pro-teins first arose in the late 1970s, with the dem-onstration3 that conjugation of multiple copies of a relatively low-molecular-weight, water-

soluble, nonionic polymer, poly(ethylene glycol) (PEG), could prolong the circulation of a therapeutic enzyme. This observation led to a flurry of activity in the ‘PEGylation’ of protein drugs, several of which have now entered the marketplace4–6.

An example that illustrates both the ben-efits and the complexities of PEGylation is interferon-α2a. The drug has been grafted at amine groups on lysine amino-acid residues to a branched, 40-kilodalton PEG chain. Although the protein is grafted with only one polymer chain, the chain can be attached to any one of four sites; as such, the drug is a mixture of four isomers. Grafting increases the hydro-dynamic radius, making the drug bulkier to promote longer retention in the circulation7. PEGylated interferon-α2a is a very successful drug for treating chronic hepatitis C.

Polymer grafting to protein drugs is associ-ated with many complexities, however, which Chilkoti and colleagues target in their work2. One such complexity, as mentioned above, is the possibility of multiple sites of polymer con-jugation, leading to a heterogeneous product. A second results from limitations in the size of the polymer chain that can be grafted. Just as it is difficult to find the end of a long rope piled up in a heap, it is difficult to graft the ter-minus of a long polymer to the surface of a pro-tein. An alternative approach, which is being

Figure 1 | Drug modification with a feather boa. Chilkoti and colleagues’ approach2 involved growing a long polymer chain from the carboxy terminus of a model protein (green fluorescent protein). The polymer unit used by the authors was oligo(ethylene glycol) methyl ether methacrylate. It is difficult to attach a long polymer chain to the end of a protein, because the two reactive sites only rarely find each other. But a chain much larger than the protein itself can be readily grown by polymerization.

Polymer units

Polymer chain

Proteina

b

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The microvasculature of tumours is known to be leaky compared with that of most normal tissues, and this difference has been used for tar-geting polymer-conjugated drugs — they leak slowly from healthy microvessels, but quickly from the microvessels supplying tumours5. However, if the drug does not circulate for long, it has little time to leak from the tumour micro-vessels. The 20-nm polymer–protein conjugate did indeed experience longer circulation times, and it leaked into tumours in mice 50 times more efficiently than the unmodified protein2. For human treatments, it would be necessary to engineer an actual drug with the polymer graft, rather than a model protein as used here, and to

show that the terminally attached polymer does not disrupt drug function.

Therapeutic proteins have already had a tremendous impact on human health, and protein–PEG conjugates make up a substantial minority of them. But the simultaneous engi-neering of the protein for the polymer and the polymer for the protein — as implemented by Chilkoti’s team — will open further doors for developing protein drugs. ■

Jeffrey A. Hubbell is at the Institute of Bioengineering and the Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL),

b I O G E O C H E M I S T R y

Phosphorus and the gust of fresh air Evidence of intense phosphorus weathering following ‘snowball Earth’ glaciations raises a further possibility — that this revved-up nutrient cycle drove conditions for the explosion of animal life. See Letter p.1088

G A b R I E L M . f I L I P P E L L I

The rapid diversification of animal (metazoan) life that started at the end of the late Proterozoic eon, about

700 million years ago, marked a turning point in Earth’s biological systems. The increase in atmospheric oxygen that occurred around this time certainly had something to do with this, by providing adequate oxidant for respiration and a sufficient stratospheric ozone layer for protection from ultraviolet radiation1,2. But perhaps the end of the widespread ‘snowball Earth’ glaciations, which covered most of the land surface and oceans in ice, may have been a factor3. Could the rapid increase in atmos-pheric oxygen be somehow related to these glaciations?

In this issue (page 1088), Planavsky et al.4 exploit analyses of the ratio of phosphorus to iron in ancient marine iron-oxide deposits to point an accusatory finger at phosphorus as the connecting agent. This element dictates global biological productivity, and con-sequently the burial of organic carbon in the ocean on geological timescales5. Past attempts to constrain the history of the phosphorus cycle have been hampered by a lack of compre-hensive understanding of phosphorus weath-ering, transport, recycling and ultimate burial in marine sediments. Recent work6–8, however, has bolstered confidence that the sedimentary record of phosphorus can be used to interpret changes in biological productivity and global phosphorus cycling on geological timescales.

Snowball EarthGlacial weathering engine Elevated atmospheric oxygen

High phosphorus input

PhotosynthesisPhosphorus + CO2 + H2O = Organic carbon + O2

High long-term organic-carbon burial sustains

oxygen addition

Figure 1 | Snowball Earth glaciations and the rise in oxygen levels around 700 million years ago4. Increased chemical weathering of terrestrial phosphorus resulted from the physical weathering caused by glacial processes. This revved-up phosphorus cycle was sustained by the lack of soil formation owing to the absence of plant rootedness, and meant that phosphorus bled off the land in bioavailable forms instead of being trapped in organically and oxide-bound forms. The continual high input of phosphorus, a biolimiting nutrient, drove intensified production of organic matter. The ensuing enhanced burial of organic carbon in turn drove addition of oxygen to the ocean and the atmosphere through their mutual long-term mass balances9 — so, speculatively, providing conditions that were ultimately conducive to metazoan evolution.

phosphorus/iron ratio that is primarily related to the phosphorus concentration of the pre-cipitating fluid. The process is also influenced by competing elements such as silicon, how-ever, and translating beaker-scale experi-ments into deep time is problematic, requiring careful selection of candidate rocks and anal-ysis of various post-depositional factors that could destroy the integrity of the phospho-rus/iron relationship. The authors mined the literature and performed analyses of their own, and then removed the silicon signal by making assumptions about past marine silicate concentrations drawn from palaeo-ecological reconstructions.

What Planavsky et al. found was remark-able — relatively ‘normal’ and constant phosphorus concentrations from 3 billion to 1.5 billion years ago, then (following a signifi-cant data gap) a huge peak reaching ten times

Planavsky et al.4 have now used the phos-phorus/iron ratio in iron-oxide-rich sedi-mentary rocks to constrain estimates of the dissolved phosphorus concentration in ancient sea water. In the laboratory, iron oxides and phosphorus co-precipitate with a characteristic

CH-1015 Lausanne, Switzerland. e-mail: jeffrey.hubbell@epfl.ch

1. Leader, B., Baca, Q. J. & Golan, D. E. Nature Rev. Drug Discov. 7, 21–39 (2008).

2. Gao, W., Liu, W., Christensen, T., Zalutsky, M. R. & Chilkoti, A. Proc. Natl Acad. Sci. USA 107, 16432–16437 (2010).

3. Abuchowski, A., McCoy, J. R., Palczuk, N. C., van Es, T. & Davis, F. F. J. Biol. Chem. 252, 3582–3586 (1977).

4. Duncan, R. Nature Rev. Drug Discov. 2, 347–360 (2003).

5. Duncan, R. Nature Rev. Cancer 6, 688–701 (2006).6. Veronese, F. M. & Pasut, G. Drug Discov. Today 10,

1451–1458 (2005).7. Bailon, P. et al. Bioconj. Chem. 12, 195–202 (2001).8. Gao, W. et al. Proc. Natl Acad. Sci. USA 106,

15231–15236 (2009).

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