l‐DNA Duplex Formation as a Bioorthogonal Information Channel in Nucleic Acid‐Based Surface Patterning

Abstract Photolithographic in situ synthesis of nucleic acids enables extremely high oligonucleotide sequence density as well as complex surface patterning and combined spatial and molecular information encoding. No longer limited to DNA synthesis, the technique allows for total control of both chemical and Cartesian space organization on surfaces, suggesting that hybridization patterns can be used to encode, display or encrypt informative signals on multiple chemically orthogonal levels. Nevertheless, cross‐hybridization reduces the available sequence space and limits information density. Here we introduce an additional, fully independent information channel in surface patterning with in situ l‐DNA synthesis. The bioorthogonality of mirror‐image DNA duplex formation prevents both cross‐hybridization on chimeric l‐/d‐DNA microarrays and also results in enzymatic orthogonality, such as nuclease‐proof DNA‐based signatures on the surface. We show how chimeric l‐/d‐DNA hybridization can be used to create informative surface patterns including QR codes, highly counterfeiting resistant authenticity watermarks, and concealed messages within high‐density d‐DNA microarrays.

Abstract: Photolithographic in situ synthesis of nucleic acids enables extremely high oligonucleotide sequence density as well as complex surfacep atterning and combined spatial and molecular information encoding.N o longer limitedt oD NA synthesis, the technique allows for total controlo fb oth chemical and Cartesian space organization on surfaces, suggesting that hybridizationp atterns can be used to encode, displayo re ncrypti nformatives ignals on multiple chemically orthogonal levels. Nevertheless, cross-hybridization reduces the available sequence space and limits information density.H ere we introduce an additional,f ully independent information channel in surface patterning with in situ l-DNA synthesis. The bioorthogonalityo fm irror-image DNA duplex formation prevents both cross-hybridization on chimeric l-/d-DNA microarrays and also resultsi ne nzymatic orthogonality,s uch as nuclease-proof DNA-based signatures on the surface. We show how chimeric l-/d-DNA hybridization can be used to create informative surface patterns including QR codes, highly counterfeiting resistanta uthenticity watermarks, and concealedm essages within high-density d-DNA microarrays.
Oligonucleotide microarrays are versatile analytical tools where very large numberso fu nique sequences are immobilizeda t precise locations on ap lanar surface to allow simultaneous access. Originally developed as platforms for gene expression analysiso fc ell populations, [1] microarrays have recently found new applicationsi ns patial transcriptomics, [2] spatialo rganization of cell-free geneticc ircuits, [3] the generation of large oligonucleotide libraries for genomic applications, [4] DNA circuitry, [5] and others. In situ synthesized microarrays yield the highest oligonucleotide sequence density and, as such, are becoming an ideal source fort he digitale ncoding of information in DNA. [6] In addition, such array fabrication offers completec ontrol over the spatial arrangement of sequences, suggesting that informative surfacep atterns may be created through simpleh ybridization-based assays. [7] Concomitant with the increasingthroughput in DNA array synthesis and the decreasing costs of sequencing, there is greater access to DNA-basedi nformation,w hich raises the potential question of privacy and traceability.I tm ay thus soonb ecome an ecessity for data stored in nucleic acid format to provide an encryption layer or atraceability signature that is only availabletothe manufacturer and customer/operator.S uch ak ey or signature could be produced in the form of binary matrices on the array itself and revealed via simple hybridization-based assays, where 0 = no hybridization and 1 = duplexf ormation with ad ye-labelled complementary probe.I deally,t his key should be synthesized alongside the bulk information,b ut not interfere with it. We have recently expanded the method of masklessa rray synthesis (MAS) [8] beyondn ative DNA, allowing for in situ synthesis of complex sequences containing 2'F-ANA [9] and RNA [10] monomers, at high densities. However exotic, these nucleic acids are nonorthogonal to cross-hybridization. While this can be mitigated by designing probes with lows equence similarity,t emperaturea nd salt concentrations can be tuned to force partial recognition.O ur search for at ruly orthogonal method that would not only preventi nteraction with standard DNA but also provide an independently accessible information channel on the array led us towards mirror-image DNA, the enantiomer of natural d-DNA.
The d-a nd l-DNA oligonucleotides of the same sequence have been shown to share common stability ands olubility characteristics [11] but differ in chirality,r esultingi nt he formation of left-handed B-form duplexes form irror-image DNA compared to the right-handedh elical conformationi nd-DNA. [12] Contradicting early reports regarding l-DNA as ap otentiala gent in antisense therapy, [13] ak ey distinctive feature in l-a nd d-forms is that hybridization exclusively occursb etween oligonucleotide strandsofequal chirality,eliminating the possibility of hybrid l-/d-DNAd uplex formation. [14] The absence of mirror-image DNA in natural biological systems seems closely related to its increased stability against DNA-degrading enzymes, [15] which is an especially appealing feature of the use of l-oligonucleotides in complex biological matrices. [16] The bioorthogonality of mirror-image oligonucleotides is indeed the basis for multiple applications, including the use of l-DNA probesi nP CR, [17] the design of nanocarriers delivering d-DNA aptamers to cells, [18] recognition of small chiral molecules [19] and the creation of heterochiral nucleic acid circuits. [20] Whereas nucleaser esistance is ac entral component of the bioorthogonal properties of l-DNA, its inability to act as as ubstrate for natural l-polymerases [21] has hindered its use in molecular biology,d espite recent efforts allowing for some key reactions to be performed using engineered d-enzymes. [22] As of ar unexploredf ield for l-DNA is in the storageo fi nformation.W hile data stored within DNA sequences can only be retrieved via sequencing, arrays of oligonucleotides allow for information to be communicated in the form of two-dimensional binary grids upon hybridization with complementary labelled probes. The scale of MAS is determined by the number of digital micromirrors, and ranges from XGA (786 432 mirrors) to 4K (8 847 360 mirrors), each mirror corresponding to ap ixel where oligonucleotide synthesis can take place. Incorporating l-DNA phosphoramidites in the process of photolithographic in situ synthesis introduces an additional information channel, which does not interferew ith d-DNA and which may be independently accessed. For these reasons, we intended to show how l-DNA synthesis, alongwith d-DNA synthesis performed in parallel (Figure 1a), can serve to label surfaces with QR codes and watermarks for authentication, or to hide messages using steganography.
Initial experiments aimed to assess and evaluate coupling time, [23] photolysis efficiency [24] and stepwise coupling yield [10a, 25] of the 5'-nitrophenylpropyloxycarbonyl (NPPOC) protected l-DNA phosphoramidites (Figure 1b), using Cy3-labelled l-a nd d-DNA complementary probes generated on separate microarrays ( Figure S1). We found that ac oupling time of 60 seconds resulted in a3 0% higherh ybridizations ignal relative to a1 5seconds coupling time. Determining the light dose required for 95 %r emoval of the photolabile protecting group revealed ad elayed photolysis of the NPPOC for l-DNA monomers compared to their d-DNA counterparts, requiring roughly 40 %h igher light exposure to yield equal photodeprotection efficiency ( Figure S3). Then, we measured the stepwise coupling efficiencies of each of the four l-a nd d-monomers (5'-NPPOC and 5'-BzNPPOC-protected,r espectively). The results, shown in Ta ble 1, indicatec omparable couplingy ields for corresponding l-/d-monomers.
Next, we wanted to examinet he fundamental differences in the biophysical properties of l-a nd d-DNA synthesized in situ on microarrays. To do so, we investigated specificity of hybridization as well as susceptibility towards an endonucleaseb y synthesizing the l-a nd d-version of the same 25-meri np arallel. First, two individual subarrays were hybridized with either an l-o rd-DNA complement. Figure 1c shows hybridization taking place highly specifically to oligonucleotides of the corresponding chirality,w ith only background fluorescencel evels for l-/d-chimeric hybrids, indicating that d-a nd l-oligonucleotides of the same sequence do not interactw ith one another, which supports the restriction to homochiral duplex formation and which was previously reported on with mixed, spotted land d-oligonucleotide arrays. [15c] Since melting temperatures of homochiral l-DNA duplexes have been shown not to differ significantly from those of natural DNA of the same sequence, [11, 15c, 19] the difference in signal intensity can be attributed to variations in purity and labelling efficiencyo ft he two probes. We then studied the resistanceo fl-DNA against nucleases   Figure 1d). First, the l-a nd d-sequences were hybridized to their complementary strands of similarc hirality (Hybridization 1, Figure 1e,l eft). The l-a nd d-duplexes were then subjected to degradation using TURBO DNase, followed by rehybridization to am ixture of complementary enantiomers( Hybridization 2). Upon DNase treatment, all d-DNAo ligonucleotides were degraded, as signaled by the complete loss of hybridization fluorescence on d-DNA features whereas l-DNA duplexes remain bright (Figure 1e,m iddle).T he rehybridization step revealed clear l-feature fluorescenceo nly,s howing that l-DNA is not affected by the nuclease, whereas the fluorescencef or d-DNA features dropped by 98 %, to background level, as expected (Figure 1e,right, and 1d). These results validate the hybridization specificitya nd complete nucleolytic resistanceo fl-DNA molecules when synthesized in situ on microarrays but, importantly,t hey show that d-DNA synthesis can be performed alongside and become an "erasable" trace among "indelible" loligonucleotides.
With no heterochiral hybridization taking place on the array and with mirror-image DNA sequences withstandingn ucleolytic treatment, we then applied l-DNA in situ synthesis fort he creationo fi nformatives urface patterns in three different contexts. In af irst application, aQ Rc ode for ar andom 128 bit key made of l-DNA was superimposed on a d-DNA pattern.R estriction of duplex formation to homochiral complementsr esulted in the l-DNA code remainingi nvisible upon hybridization solely with a d-DNA probe. After addition of the l-DNA probe however,t he code appears ( Figure 2a)a nd resists endonucleolytic degradation (Figure 2b).
Following our first attempts at producing informative, l-DNA-basedp atterns, we then generated d-DNA microarrays supplemented with an l-DNA authenticity watermark as ap otential signature for microarrays originating from our laboratory.W ef ollowed an encryption schemef or oligonucleotide microarrays recently developed by Holden et al. [26] The approach prevents af orger from deciphering as equence using sequenc-ing by hybridization( SBH), which is the only methoda llowing for sequence information to be retrievedw hile retaining the spatialo rdering of oligonucleotide strandso nt he substrate. At the core of the approach, two individual oligonucleotide strandso fh igh sequence similaritya re combined within a single pixel, thus rendering SBH signals impossible to be assignedt oo nly one of the strands. Inspired by this system, we produced the two strands/one feature combination by synthesizing two l-DNAs equences in ar ow,s pacedb yad-DNA T 5 , thus creatingasingle 3'-L x -d-L y -5' sequence insteado ft wo individual strands. The d-DNA spacer prevents sequence informationr etrieval through SBH via ad iscontinuity between the encoding strands. In ap roof-of-concept, an array of 5 5p ixels at one cornero ft he microarray was used for l-DNA synthesis to produce ad istinct signal pattern upon hybridization with the correct key probe, whereas the remaining part of the synthesis area consisted of a d-DNA 25-mer (layout shown in Figure 3a). The L x andL y sequences were generated as combinations of blocks of three specific 10-mers (namedA,Band C) according to the scheme and calculations discussed elsewhere [26] (see Table S1). Here, creating an l-DNA watermark allows for any other d-DNA sequences to be addressed withoutt he risk of interference with the signature. To create truly undecipherable 2D patterns,f ive different combinationso fL x -d-L y chimeras were designed (V1 to V5, setup according to Figure 3c), resulting in the pattern shown in Figure 3b upon hybridization with as ingle labelled l-DNA probe (L ABC-complement ). An additional level of intensity is created through the introduction of background features (BG).
These complexw atermarks would be particularly labor-intensivetoimitate because of three obstacles:sequence similarity between L x and L y preventing SBH, combinations of ABC blockst ow hich ag iven probe may or may not hybridize, and non-hybridizedf eatures being equivalent to background.F urthermore, l-DNA sequence identityc annot be recovered by cleaving andi solating the l-oligonucleotides,e ven after sacrificing spatiali nformation, since current high-throughput se-  Finally,w ea pplied l-DNA in steganography as aw ay to conceal am essage within ap hotographic reproduction composed in d-DNA with ar esolution of 1024 768 pixels. The message is encoded in decimal form on the x coordinates of l-DNA pixels. The premise of the approachi sb ased on the assumption that af ew additional features lighting up would seem inconspicuous to the naked eye, yet would be identifiable by standard data extraction.T he pattern visible after initial hybridization with ac omplementary d-DNA probe indeed does not suspiciously differ from the version after hybridization with am ix of d-a nd l-DNA probe (Figure 4cd). Aligning scans with the underlyingm icroarray design followed by data analysis allows for the coordinates of the pixels displaying unusual florescence to be recognized. The hiddenm essage (Figure 4e and Table S2) can then be retrieved using an ASCII table.
In summary,w ep resented the additiono fl-DNA phosphoramiditest oo ur toolbox of buildingb locks availablef or photolithographic in situ synthesis of microarrays.W es how that the biophysical properties of mirror-imageD NA,i ncluding homochiral hybridization behavior and increased nuclease stability remain valid for microarray-synthesized oligonucleotides. The fluorescently labelled probesr equiredf or on-array hybridization are generated on as eparate microarray,c leaved and retrieved in solution, whicho pens the way to the preparation of large l-DNA libraries.Wethen explore an ew avenue for l-DNA as ab ioorthogonal hybridization tool in the creationo ft wo-dimensionalb inary patternsc ontaining authentication and encryptedm essages. Chimeric l-/d-DNA microarrays can thus form two independent information channels that can each be accessed separately by hybridization to fluorescently labelled probes. Within standard d-DNA oligonucleotide arrays, l-DNA features were designed to form QR codes on the array that may reveal synthesis data as well as provide decoding keys for encrypted information stored on d-DNA. Forgery-proof l-DNA watermarks can be used to confirma uthenticity,a nd sensitive data can be concealed as code in the coordinates of complex synthetic array patterns. The use of mirror-imageo ligonucleotides in these applications as add-ons to common microarrays does not only offer an additional level of pure synthetic complexity, but the clear bioorthogonality between l-a nd d-enantiomers also brings the prospectf or parallelized assays to be performedo ns urface-bound l-/d-oligo libraries, such as in DNA-based logic circuits.