© 2003 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003

15.3.6.1 Biocompatibility of DNA

Seeman [2247, 5666-5668] has pioneered the exploration of DNA as a nanoscale construction material* (Section 2.3.1), and a few others have employed DNA in related contexts [6180-6182], raising the question of the biocompatibility of structural nucleotides that might be used to build early-generation nanomedical devices. Other popular variants on DNA such as peptide nucleic acid molecules [5664] might also find utility in nanomedicine and genetic therapies [5665], and metal-containing DNA [6019] or metallo-DNA could be used as nanocomputer wiring and thus might be found inside the body upon accidental fragmentation (Section 15.4.4, Chapter 17) of nanorobots using this type of circuitry.

* It has been proposed that the mechanical properties of DNA may have contributed to the ability of early cellular life (protocells) to withstand turgor pressure [5979].

DNA in its natural helical state (dry bulk DNA density ~1.65 gm/cm³) [6249] is usually nonimmunogenic in normal animals [1760]. Ordinary DNA placed in human serum is degraded rapidly by natural nuclease enzymes (plasma nucleases); post-apoptotic nuclear DNA is depolymerized and opsonized by serum factors [2248]. IgG in human blood serum [2250] and in human milk [2251] can hydrolyze both DNA and RNA. Nucleases in human blood serum can degrade double-stranded DNA, RNA and synthetic polyribonucleotides [2252], antisense oligonucleotides [2253], and various oligodeoxynucleotides [2254]. Single-stranded (ss) oligonucleotides are more susceptible to hydrolysis than double-stranded (ds) oligonucleotides [2254]. High molecular weight ssDNA is rapidly cleaved to 20-30 kD fragments by endonucleases, with mononucleotide breakdown products appearing in circulation with no lag time [2368]. The short half-life of DNA can be exploited for use with certain therapeutics that require moment-to-moment control, especially aptamers that have been developed to alter blood coagulation [5801-5807] or other biologic processes [5808-5810]. Chemical modifications [2255-2257] can improve the resistance of oligonucleotides – especially antisense oligonucleotides [4698-4719] – to nuclease attack. These methods might prove useful in serum-proofing early DNA-based medical nanodevices [5097]. However, some of these changes cause the synthetic material to become toxic in vivo, so every form of chemically modified nucleic acid will have to be extensively evaluated in animal and human toxicity testing.

The physiological response to free DNA may be relevant to bloodborne DNA-based nanodevices. The normal concentration of free DNA in human serum is very low [2369], typically 5-40 ng/ml [2370]. Free fetal DNA is found in maternal plasma [2371] at 0.2 ng/ml (range 0.03-0.6 ng/ml) in early pregnancy, 0.6 ng/ml in midterm pregnant women, 2 ng/ml (range 0.6-6 ng/ml) in late pregnancy, 3 ng/ml in preeclamptic women [2370, 2372], and then falling to near undetectable levels 2 hours postpartum [2373]. Circulating DNA is found in patients with (1) autoimmune thyroid disorders [2374]; (2) pulmonary embolism [2375]; (3) systemic arterial inflammation or vasculitis (20-50 ng/ml plasma dsDNA) [2376]; and (4) neoplasms of various types such as benign gastrointestinal lesions (118 ng/ml) [2377] or malignant disease (412 ng/ml) [2377]. It is also found in patients during hemodialysis (up to 5000 ng/ml plasma DNA) [2378], presumably due to release from leukocytes, and DNA plasma concentrations as high as 16,000 ng/ml (~50 billion micron³, in whole human bloodstream) have been recorded (Appendix B). The mean half-life for circulating fetal DNA in maternal plasma is 16 min (range 4-30 min) [2373]. Clearance of free dsDNA has a half-life of 18 min in nonhuman primates, or 11 min for immune-complexed (IC) dsDNA [2379]. Up to 85% of IC-dsDNA (typically IgG [2380]) binds to erythrocyte surfaces within 2 min of injection [2379]. The liver is the primary uptake site [2369, 2379]. Organ uptake is more rapid for ssDNA than for dsDNA [2368, 2381]. DNA larger than 15 bp does not measurably persist in the mouse bloodstream longer than 20 min for ssDNA, or 40 min for dsDNA [2381]. At high doses the clearance rate reaches a maximum, allowing larger amounts of ssDNA to remain in circulation [2368].

Anti-DNA antibodies are found in normal human subjects [2258] and in the sera of patients with some autoimmune diseases such as systemic lupus erythematosis (SLE) [2259], catastrophic or even asymptomatic/remission antiphospholipid syndrome (APS) [5392], or thyroid disorders [2374]. SLE patients produce anti-DNA that targets conserved sites on both ssDNA and dsDNA from essentially all species [2260], with anti-dsDNA antibodies possibly recognizing unique structures around the G+C regions or G+C clusters of DNA [2261] and binding preferentially to poly(dA-dC) and poly(dG-dT) [2267]. In normal subjects without SLE, the serum only contains anti-DNA antibodies that selectively bind to DNA from certain bacteria [2260], but native DNA mutated by UV light and hydrogen peroxide has been rendered immunogenic in experimental animals [2262]. Bacterial DNA is a potent mitogen and immunogen. Immunization of normal animals with bacterial DNA elicits antibodies that bind mammalian as well as bacterial ssDNA, and also induces cytokine production in the mouse [2260] and can produce other immunostimulatory effects depending on methylation [5811-5813].

Solid-phase binding of DNA segments (as might occur in DNA-based medical nanomachines) dramatically reduces DNA antigenicity because constraints on topological and conformational rearrangements of DNA in the solid phase hinder antibody [2263] and nuclease (a potentially confounding issue) interactions. The length of these DNA segments appears unimportant, at least in undiseased humans [2264]. Antibodies can recognize B-DNA [2269], A-DNA/RNA hybrids [2269], and even the left-handed Z-DNA [2265-2269] found in some of Seeman’s earlier experimental structures. (In 2002, the most promising structures appeared to be DNA-based PX-JX2 devices [5666] that used no Z-DNA [Nadrian C. Seeman, personal communication, 2002]; antibody recognition of these new structures had not yet been reported.) The usual risk of insertional mutagenesis from nucleic acid medicines [2270] should be greatly reduced in DNA-based nanodevices as long as these nanomachines remain intact. Biological activity (translational, enzymatic, etc.) of artificial DNA sequences comprising DNA-based devices or their fragments cannot be ruled out and should be investigated in every case; such activity is most likely to occur in devices having components specifically designed for biochemical interaction, or having sequences derived from natural templates (e.g., viral, bacterial, mammalian). (Infective naked viral DNA should not be considered “biocompatible.”) DNA-coated charcoal granules and carbon fibers have shown good biocompatibility [2271], and some synthetic oligonucleotides actually inhibit coagulation and reduced hemolytic complement activity in vitro [2272, 2273], an effect which appears to be nucleotide sequence-independent [2273] as mentioned above.

As with many cell types, keratinocytes [5576] can take up oligodeoxynucleotides and plasmid DNA, probably by receptor-mediated endocytosis, inducing the production of interleukin (IL)-1alpha, IL-1beta, integrin-beta(1), alpha-tubulin, and follistatin. Free deoxynucleoside in concentrations of 2-5 mM is well tolerated by living cells experimentally [5577]. As for free DNA released intracellularly, leukocytes contain nucleases that break down ingested DNA [2249], and intracellular nucleic acids are starting to be studied for their possible diagnostic value [5578]. Intracellular nucleases are known for DNA [5579] and RNA [5580-5583] degradation, and especially for mRNA [5584-5589] degradation wherein the degradation process is initiated by deadenylation [5601] and is tightly regulated [5589-5592]. Both single- and double-stranded circular plasmid DNA is degraded in ~1 hour by cytosolic nucleases [4295, 4305, 4306]. It should also be noted that apoptotic cells (Section 10.4.1.1) degrade their DNA before it is released, preventing inflammatory responses.

Double-stranded RNA (e.g., ~500 nucleotides in length [5972]) can induce the degradation of homologous mRNAs in organisms as diverse as protozoa, animals, plants and fungi, and especially mammals [5972], resulting in post-transcriptional gene silencing (termed RNA interference or RNAi) [5973, 6016] that takes place only in the cytoplasm [5974]. The dsRNAi is itself degraded in the cell [5975]. Apparently RNA interference reflects an elaborate cellular apparatus that eliminates abundant but defective mRNAs and defends against molecular parasites such as transposons and viruses [5976]. The recently discovered process of DNA-RNA interference [5977] suggests that cells are very sensitive to double-stranded DNA or RNA – which is apparently misinterpreted as a viral infection, causing cells to enter viral defense mode and/or turn off those genes that are producing dsDNA/RNA. Even mRNA-cDNA hybrid constructs can produce relatively long-term interference of specific gene expression [5978]. Nanorobots using nonhomologous DNA/RNA sequences in the cytosol-accessible portion of their structures should not elicit these cellular responses.

Last updated on 30 April 2004