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(Task 3) APPLICATIONS: RECENT PROGRESS and ACCOMPLISHMENTS Sept. 1, 1998-April 30, 1999 (3.1) DNA Cryptography and Steganography Recent research has considered DNA as a medium for ultra-scale computation and for ultra-compact information storage. One potential key application is DNA-based, molecular cryptography systems. Since this work constitutes a novel approach to the use of DNA in the area of cryptography, it is expected to be of significant interest to the military and thus to DARPA. We developed some procedures for DNA-based cryptography based on one-time-pads that are in principle unbreakable. Practical applications of cryptographic systems based on one-time-pads are limited in conventional electronic media, by the size of the onetime-pad; however DNA provides a much more compact storage media, and an extremely small amount of DNA suffices even for huge one-time-pads. We detailed procedures for two DNA one-time-pad encryption schemes: (i) a substitution method using huge libraries of distinct pads, each of which defines a specific, randomly generated, pair-wise mapping; and (ii) an XOR scheme utilizing molecular computation and indexed, random key strings. These methods can be applied either for the encryption of (appropriately recoded) natural DNA, and also for the encryption of DNA encoding binary data. In the latter case, we also present a novel use of chip-based DNA micro-array technology for 2D data input and output. (Note: The above LaBean, et al paper also critically examined a large class of DNA steganography systems, and showed that in certain cases such schemes may not cryptographically secure, and can be broken with certain assumptions on the entropy of the plaintext messages.) Gehani, A., T. H. LaBean, J. H. Reif, DNA-based Cryptography, 5th International Meeting on DNA Based Computers(DNA5), MIT, Cambridge, MA, (June, 1999). To appear in DIMACS Series in Discrete Mathematics and Theoretical Computer Science, ed. E. Winfree, (1999). www.cs.duke.edu/~reif/paper/DNAcrypt/crypt.ps Graphic illustrations of DNA Cryptography: http://www.cs.duke.edu/~reif/paper/DNAcrypt/crypt.talk/crypt.talk.web/crypt.talk.html http://www.cs.duke.edu/~reif/paper/DNAcrypt/crypt.talk/crypt.talk.pdf DNA Steganography The work is in a sub-category of cryptography, called steganography, and involves hiding a (possibly encrypted) message in such a way that an observer (the "enemy") cannot detect or read it. We investigated DNA steganography systems, which secretly tag the input DNA and then disguise it (without further modifications) within collections of other DNA. The below paper developed and executed experimentally a novel approach to using a DNA steganographic technique in which a DNA-encoded message is hidden within human genomic DNA. Advantage is taken of the enormous complexity of the genomes of multi-cellular organisms (we have employed the human genome as an example) to hide a secret DNA- encoded message in the midst of this complexity. A text message is encoded into DNA according to some simple encryption scheme (in this simplest model case, it is not necessary that this encoding be difficult to crack; both because encryption is not the major basis for keeping the enemy from reading the secret text, and because the enemy is very unlikely to ever see the secret message and thus will not even have an opportunity to attempt to break the code employed for encryption). A "Secret Message" (SM) DNA molecule is then synthesized containing three parts: the secret message in the middle, flanked on each side by specific primer sequences known only to the sender ("Alice") and the recipient ("Bob"). A small amount of the SM DNA is then added to an appropriately treated human DNA sample, and sent from Alice to Bob. To read the message, Bob carries out PCR on the DNA sample he has received, employing a primer pair complementary to the two primer sequences described above. (Although this might appear similar to the classical "one-time pad" cryptographic technique, it is in fact reusable, and is thus a more generally useful procedure than the one-time pad technique, because the same primer sequences (and encryption key) could be employed on many separate occasions for communications between Alice and Bob. This is because the enemy does not know that the human DNA sample contains a secret message; more importantly, even if he did know this, he would have no way to identify the SM DNA and thus read the sequences of either the primers or the encoded message). Bob then recovers the PCR-amplified DNA product, reads the sequence of the SM DNA molecule, and then employs the simple encryption key (also agreed upon in advance by Alice and Bob) to decode and read the message that is encoded in the DNA sequence residing between the primers. Bancroft,C. C.T. Clelland, V. Risca, Genomic Steganography: Amplifiable Microdots, 5th International Meeting on DNA Based Computers(DNA5), MIT, Cambridge, MA, (June, 1999). To appear in DIMACS Series in Discrete Mathematics and Theoretical Computer Science, ed. E. Winfree, (1999). (3.1) DNA2DNA Computations We experimentally demonstrated key stages of the re-coding of natural DNA using nonstandard bases to code digital data. This experimental test is significant if we are to avoid the usual very time consuming task of sequencing natural DNA into conventional binary electronic form. The encoded DNA sequences were formed by a hybridization reaction. We made use of a thermostable DNA that allow us to amplify and capture the ligated strands by the ligase chain reaction (LCR). We tested the following features: a) the minimal length of probe strands to be ligated; b) the optimal temperature of ligation in terms of (1) efficiency of ligation, and (2) fidelity of ligation. We also developed a protocol for counting the number of different kinds of strands of DNA in the test tube. http://www.princeton.edu/~lfl/DNA2DNA/ PLANNED WORK for the rest of FY99 and FY2000 (3.1) DNA2DNA Computations We are now completing the experimental demonstration of all stages of the re-coding of natural DNA using non-standard bases to code digital data. The completion of these experimental tests of re-coding will be significant if we are to avoid the usual very time consuming task of sequencing natural DNA into conventional binary electronic form. Fingerprinting DNA: In our recent work on genetic recombination intermediates, we have discovered that it is possible to test for the presence of a particular sequence in a double helical context. This test, which does not involve proteins, appears to be independent of sequence effects. We plan to pursue this type of molecular recognition, and to develop means to incorporate it into a DNA computing context. This may lead to exquisitely sensitive methods for fingerprinting DNA. (3.2) DNA Cryptography and Steganography DNA Cryptography. We plan to perform experimental prototyping of some methods useful for cryptographic systems involving DNA, especially a novel input/output scheme which uses DNA-chips containing sizable arrays of addressable DNA sequences. DNA Steganography. We are considering experimental tests of our method for genomic steganography using amplifiable microdots. The achievement of optimal utility for this technique will require the use of solid substrates other than the filter paper we have employed for the above studies. We thus plan to attempt to adapt this approach for use with various common, apparently innocuous substrates, such as typing paper, paper currency, etc. We plan also to work on scaling down the size of the dots employed to carry the message hidden in DNA, thus decreasing the ability of an adversary to detect the presence of the DNA-encoded secret message.