**Acknowledgement**

This research was supported by the Russian Academy of Science under the programs Molecular and Cell Biology (grant no. 22-1) and Science to Medicine (grant no. 37), the Russian Foundation for Basic Research (grant nos. 11-04-01017-a and 11-04-12095-ofi-m-2011) Ministry of Science and Education of the Russian Federation (grant no. 14.740.11.1058) and the Siberian Branch of Russian Academy of Sciences (grant no. 85).

#### **6. References**

216 Practical Applications in Biomedical Engineering

strongly affected by the nature, number and locations within the duplex of chemical modifications [65, 76, 181]. Despite many works carried out in this area, a general algorithm

Chemical modification of siRNA can be also used to develop delivery vehicles for siRNA. It is well known that naked or free siRNA is not able to penetrate through cellular membrane and accumulate in the cells. In the case of in vivo conditions, the small size of siRNA molecule promotes its rapid clearance [57]. In other words, the systems for in vivo delivery of siRNAs should provide for efficiency of cellular accumulation, specificity of delivery (if applicable), duration of silencing as well as safety and possibility of systemic

On the other hand, the low efficiency of RNAi can be a result of its unfavorable sequence determining thermodynamic properties and/or by stable secondary structure at the target site within mRNA [37, 38, 51, 52, 54]. There are several algorithms to select siRNAs of highperformance [182, 183, 184, 185, 186], however it is impossible to predict precisely silencing activity of any particular siRNA in vivo. In addition, the design of siRNA targeting chimeric or mutant genes represent of complex task since the limitations of target site selection does not allow to use these algorithms for choosing the sequence siRNA. This is why structure modifications of siRNA improving their thermodynamic properties are of interest. The forklike siRNA [158, 159], able to silence target gene more efficiently that conventional siRNA is a promising approach for creating high-performance siRNA targeted any sequence of the gene. Despite this, the number of works in this field is negligible so further research of

To obtain high-performance siRNA often optimization of multiple parameters is required, so using modification of the siRNA structure together with its chemical modification is a promising approach. Thus, advanced research of chemically and/or structurally modified siRNA, as well as development and optimization of the algorithms for modification are

This research was supported by the Russian Academy of Science under the programs Molecular and Cell Biology (grant no. 22-1) and Science to Medicine (grant no. 37), the Russian Foundation for Basic Research (grant nos. 11-04-01017-a and 11-04-12095-ofi-m-2011) Ministry of Science and Education of the Russian Federation (grant no. 14.740.11.1058)

which allows receiving active nuclease resistant siRNA is not developed yet.

administration. Today no one delivery approach encounters these criteria.

properties of siRNA with modified structure are relevant.

Natalya S. Petrova, Marina A. Zenkova and Elena L. Chernolovskaya

and the Siberian Branch of Russian Academy of Sciences (grant no. 85).

*Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia* 

important.

**Author details** 

**Acknowledgement** 


[21] Ma J. B., Ye K. and Patel D. J. (2004) Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature. 429, 318-322.

Structure - Functions Relations in Small Interfering RNAs 219

[40] Elbashir S. M., Martinez J., Patkaniowska A., Lendeckel W. and Tuschl T. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila

[41] Liu J., Carmell M. A., Rivas F. V., Marsden C. G., Thomson J. M., Song J. J., Hammond S. M., Joshua-Tor L. and Hannon G. J. (2004) Argonaute2 is the catalytic engine of

[42] Braasch D. A. and Corey D. R. (2002) Novel antisense and peptide nucleic acid

[43] Dorsett Y. and Tuschl T. (2004) siRNAs: applications in functional genomics and

[44] Bertrand J. R., Pottier M., Vekris A., Opolon P., Maksimenko A. and Malvy C. (2002) Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo.

[45] Elbashir S. M., Lendeckel W. and Tuschl T. (2001) RNA interference is mediated by 21-

[46] Prakash T. P., Allerson C. R., Dande P., Vickers T. A., Sioufi N., Jarres R., Baker B. F., Swayze E. E., Griffey R. H. and Bhat B. (2005) Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J Med Chem. 48, 4247-4253. [47] Chu C. Y. and Rana T. M. (2006) Translation repression in human cells by microRNA-

[48] Jakymiw A., Pauley K. M., Li S., Ikeda K., Lian S., Eystathioy T., Satoh M., Fritzler M. J. and Chan E. K. (2007) The role of GW/P-bodies in RNA processing and silencing. J Cell

[49] Chalk A. M., Wahlestedt C. and Sonnhammer E. L. (2004) Improved and automated

[50] Reynolds A., Leake D., Boese Q., Scaringe S., Marshall W. S. and Khvorova A. (2004)

[51] Brown K. M., Chu C. Y. and Rana T. M. (2005) Target accessibility dictates the potency

[52] Gredell J. A., Berger A. K. and Walton S. P. (2008) Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study. Biotechnol Bioeng. 100, 744-755. [53] Holen T., Amarzguioui M., Wiiger M. T., Babaie E. and Prydz H. (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.

[54] Westerhout E. M. and Berkhout B. (2007) A systematic analysis of the effect of target

[55] Amarzguioui M., Brede G., Babaie E., Grotli M., Sproat B. and Prydz H. (2000) Secondary structure prediction and in vitro accessibility of mRNA as tools in the

[56] Braasch D. A., Paroo Z., Constantinescu A., Ren G., Oz O. K., Mason R. P. and Corey D. R. (2004) Biodistribution of phosphodiester and phosphorothioate siRNA. Bioorg Med

[57] Soutschek J., Akinc A., Bramlage B., Charisse K., Constien R., Donoghue M., Elbashir S., Geick A., Hadwiger P., Harborth J., John M., Kesavan V., Lavine G., Pandey R. K., Racie T., Rajeev K. G., Rohl I., Toudjarska I., Wang G., Wuschko S., Bumcrot D., Koteliansky

prediction of effective siRNA. Biochem Biophys Res Commun. 319, 264-274.

Rational siRNA design for RNA interference. Nat Biotechnol. 22, 326-330.

RNA structure on RNA interference. Nucleic Acids Res. 35, 4322-4330.

selection of target sites for ribozymes. Nucleic Acids Res. 28, 4113-4124.

strategies for controlling gene expression. Biochemistry. 41, 4503-4510.

potential as therapeutics. Nat Rev Drug Discov. 3, 318-329.

induced gene silencing requires RCK/p54. PLoS Biol. 4, e210.

melanogaster embryo lysate. Embo J. 20, 6877-6888.

mammalian RNAi. Science. 305, 1437-1441.

Biochem Biophys Res Commun. 296, 1000-1004.

and 22-nucleotide RNAs. Genes Dev. 15, 188-200.

of human RISC. Nat Struct Mol Biol. 12, 469-470.

Nucleic Acids Res. 30, 1757-1766

Chem Lett. 14, 1139-1143.

Sci. 120, 1317-1323.


[40] Elbashir S. M., Martinez J., Patkaniowska A., Lendeckel W. and Tuschl T. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J. 20, 6877-6888.

218 Practical Applications in Biomedical Engineering

21, 5875-5885.

434, 666-670.

Mol Biol. 12, 340-349.

Cell. 123, 607-620.

RNA. Nature. 431, 343-349.

[21] Ma J. B., Ye K. and Patel D. J. (2004) Structural basis for overhang-specific small

[22] Zhang H., Kolb F. A., Brondani V., Billy E. and Filipowicz W. (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. Embo J.

[23] Grunweller A. and Hartmann R. K. (2005) RNA interference as a gene-specific approach

[24] Kini H. K. and Walton S. P. (2009) Effect of siRNA terminal mismatches on TRBP and

[25] Liu X., Jiang F., Kalidas S., Smith D. and Liu Q. (2006) Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. Rna. 12, 1514-1520. [26] Hutvagner G. and Simard M. J. (2008) Argonaute proteins: key players in RNA

[27] Song J. J., Smith S. K., Hannon G. J. and Joshua-Tor L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science. 305, 1434-1437. [28] Bernstein E., Caudy A. A., Hammond S. M. and Hannon G. J. (2001) Role for a bidentate

[29] Ma J. B., Yuan Y. R., Meister G., Pei Y., Tuschl T. and Patel D. J. (2005) Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature.

[30] Parker J. S., Roe S. M. and Barford D. (2005) Structural insights into mRNA recognition

[31] Rivas F. V., Tolia N. H., Song J. J., Aragon J. P., Liu J., Hannon G. J. and Joshua-Tor L. (2005) Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct

[32] Meister G. and Tuschl T. (2004) Mechanisms of gene silencing by double-stranded

[33] Nykanen A., Haley B. and Zamore P. D. (2001) ATP requirements and small interfering

[34] Leuschner P. J., Ameres S. L., Kueng S. and Martinez J. (2006) Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314-320. [35] Matranga C., Tomari Y., Shin C., Bartel D. P. and Zamore P. D. (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.

[36] Addepalli H., Meena, Peng C. G., Wang G., Fan Y., Charisse K., Jayaprakash K. N., Rajeev K. G., Pandey R. K., Lavine G., Zhang L., Jahn-Hofmann K., Hadwiger P., Manoharan M. and Maier M. A. (2010) Modulation of thermal stability can enhance the

[37] Khvorova A., Reynolds A. and Jayasena S. D. (2003) Functional siRNAs and miRNAs

[38] Schwarz D. S., Hutvagner G., Du T., Xu Z., Aronin N. and Zamore P. D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell. 115, 199-208. [39] Haley B. and Zamore P. D. (2004) Kinetic analysis of the RNAi enzyme complex. Nat

ribonuclease in the initiation step of RNA interference. Nature. 409, 363-366.

from a PIWI domain-siRNA guide complex. Nature. 434, 663-666.

RNA structure in the RNA interference pathway. Cell. 107, 309-321.

potency of siRNA. Nucleic Acids Res. 38, 7320-7331.

exhibit strand bias. Cell. 115, 209-216.

Struct Mol Biol. 11, 599-606.

interfering RNA recognition by the PAZ domain. Nature. 429, 318-322.

for molecular medicine. Curr Med Chem. 12, 3143-3161.

silencing. Nat Rev Mol Cell Biol. 9, 22-32.

Dicer binding and silencing efficacy. FEBS J. 276, 6576-6585.


V., Limmer S., Manoharan M. and Vornlocher H. P. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 432, 173-178.

Structure - Functions Relations in Small Interfering RNAs 221

[73] Kraynack B. A. and Baker B. F. (2006) Small interfering RNAs containing full 2'-Omethylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent

[74] Hamada M., Ohtsuka T., Kawaida R., Koizumi M., Morita K., Furukawa H., Imanishi T., Miyagishi M. and Taira K. (2002) Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs. Antisense Nucleic Acid Drug Dev. 12, 301-309. [75] Parrish S., Fleenor J., Xu S., Mello C. and Fire A. (2000) Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. Mol

[76] Watts J. K., Deleavey G. F. and Damha M. J. (2008) Chemically modified siRNA: tools

[77] Allerson C. R., Sioufi N., Jarres R., Prakash T. P., Naik N., Berdeja A., Wanders L., Griffey R. H., Swayze E. E. and Bhat B. (2005) Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small

[78] Layzer J. M., McCaffrey A. P., Tanner A. K., Huang Z., Kay M. A. and Sullenger B. A.

[79] Muhonen P., Tennila T., Azhayeva E., Parthasarathy R. N., Janckila A. J., Vaananen H. K., Azhayev A. and Laitala-Leinonen T. (2007) RNA interference tolerates 2'-fluoro

[80] Ikeda H., Fernandez R., Wilk A., Barchi J. J., Jr., Huang X. and Marquez V. E. (1998) The effect of two antipodal fluorine-induced sugar puckers on the conformation and stability of the Dickerson-Drew dodecamer duplex [d(CGCGAATTCGCG)]2. Nucleic

[81] Damha M. J., Noronha A. M., Wilds C. J., Trempe J. F., Denisov A., Pon R. T. and Gehring K. (2001) Properties of arabinonucleic acids (ANA & 20'F-ANA): implications for the design of antisense therapeutics that invoke RNase H cleavage of RNA.

[82] Ui-Tei K., Naito Y., Zenno S., Nishi K., Yamato K., Takahashi F., Juni A. and Saigo K. (2008) Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing

[84] Dorn G., Patel S., Wotherspoon G., Hemmings-Mieszczak M., Barclay J., Natt F. J., Martin P., Bevan S., Fox A., Ganju P., Wishart W. and Hall J. (2004) siRNA relieves

[85] Chen X., Shen L. and Wang J. H. (2004) Poly-2'-DNP-RNAs with enhanced efficacy for

[86] Liao H. and Wang J. H. (2005) Biomembrane-permeable and Ribonuclease-resistant

[87] Ashun M. A., Hu Y., Kang I., Li C. C. and Wang J. H. (1996) Inhibition of murine leukemia virus with poly-2'-O-(2,4-dinitrophenyl)poly[A]. Antimicrob Agents

with significantly reduced off-target effect. Nucleic Acids Res. 36, 2136-2151. [83] Amarzguioui M., Holen T., Babaie E. and Prydz H. (2003) Tolerance for mutations and

chemical modifications in a siRNA. Nucleic Acids Res. 31, 589-595.

(2004) In vivo activity of nuclease-resistant siRNAs. RNA. 10, 766-771.

modifications at the Argonaute2 cleavage site. Chem Biodivers. 4, 858-873.

activity. RNA. 12, 163-176.

and applications. Drug Discov Today. 13, 842-855.

Nucleosides Nucleotides Nucleic Acids. 20, 429-440.

chronic neuropathic pain. Nucleic Acids Res. 32, e49.

inhibiting cancer cell growth. Oligonucleotides. 14, 90-99.

siRNA with enhanced activity. Oligonucleotides. 15, 196-205.

interfering RNA. J Med Chem. 48, 901-904.

Cell. 6, 1077-1087.

Acids Res. 26, 2237-2244.

Chemother. 40, 2311-2317.


[73] Kraynack B. A. and Baker B. F. (2006) Small interfering RNAs containing full 2'-Omethylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity. RNA. 12, 163-176.

220 Practical Applications in Biomedical Engineering

analysis. Rna. 9, 1034-1048.

in serum. Biochem Pharmacol. 71, 702-710.

replication. Hepatology. 41, 1349-1356.

RNAs. Curr Opin Chem Biol. 8, 570-579.

interfering RNA. Hum Gene Ther. 19, 111-124.

suppression by siRNA via TLR3. Nature. 452, 591-597.

activity. Mol Biosyst. 3, 43-50.

1-14.

Res. 28, 221-233.

7967-7975.

Commun. 342, 919-927.

V., Limmer S., Manoharan M. and Vornlocher H. P. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 432, 173-178. [58] Chiu Y. L. and Rana T. M. (2003) siRNA function in RNAi: a chemical modification

[59] Czauderna F., Fechtner M., Dames S., Aygun H., Klippel A., Pronk G. J., Giese K. and Kaufmann J. (2003) Structural variations and stabilising modifications of synthetic

[60] Haupenthal J., Baehr C., Kiermayer S., Zeuzem S. and Piiper A. (2006) Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs

[61] Morrissey D. V., Blanchard K., Shaw L., Jensen K., Lockridge J. A., Dickinson B., McSwiggen J. A., Vargeese C., Bowman K., Shaffer C. S., Polisky B. A. and Zinnen S. (2005) Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus

[62] Turner J. J., Jones S. W., Moschos S. A., Lindsay M. A. and Gait M. J. (2007) MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like

[63] White P. J. (2008) Barriers to successful delivery of short interfering RNA after systemic

[64] Judge A. D., Bola G., Lee A. C. and MacLachlan I. (2006) Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol Ther. 13, 494-505. [65] Manoharan M. (2004) RNA interference and chemically modified small interfering

[66] Robbins M., Judge A., Liang L., McClintock K., Yaworski E. and MacLachlan I. (2007) 2'-

[67] Judge A. and MacLachlan I. (2008) Overcoming the innate immune response to small

[68] Takeda K. and Akira S. (2005) Toll-like receptors in innate immunity. Int Immunol. 17,

[69] Zamanian-Daryoush M., Marques J. T., Gantier M. P., Behlke M. A., John M., Rayman P., Finke J. and Williams B. R. (2008) Determinants of cytokine induction by small interfering RNA in human peripheral blood mononuclear cells. J Interferon Cytokine

[70] Kleinman M. E., Yamada K., Takeda A., Chandrasekaran V., Nozaki M., Baffi J. Z., Albuquerque R. J., Yamasaki S., Itaya M., Pan Y., Appukuttan B., Gibbs D., Yang Z., Kariko K., Ambati B. K., Wilgus T. A., DiPietro L. A., Sakurai E., Zhang K., Smith J. R., Taylor E. W. and Ambati J. (2008) Sequence- and target-independent angiogenesis

[71] Braasch D. A., Jensen S., Liu Y., Kaur K., Arar K., White M. A. and Corey D. R. (2003) RNA interference in mammalian cells by chemically-modified RNA. Biochemistry. 42,

[72] Choung S., Kim Y. J., Kim S., Park H. O. and Choi Y. C. (2006) Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochem Biophys Res

O-methyl-modified RNAs act as TLR7 antagonists. Mol Ther. 15, 1663-1669.

siRNAs in mammalian cells. Nucleic Acids Res. 31, 2705-2716.

administration. Clin Exp Pharmacol Physiol. 35, 1371-1376.

	- [88] Bramsen J. B., Laursen M. B., Nielsen A. F., Hansen T. B., Bus C., Langkjaer N., Babu B. R., Hojland T., Abramov M., Van Aerschot A., Odadzic D., Smicius R., Haas J., Andree C., Barman J., Wenska M., Srivastava P., Zhou C., Honcharenko D., Hess S., Muller E., Bobkov G. V., Mikhailov S. N., Fava E., Meyer T. F., Chattopadhyaya J., Zerial M., Engels J. W., Herdewijn P., Wengel J. and Kjems J. (2009) A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res. 37, 2867-2881.

Structure - Functions Relations in Small Interfering RNAs 223

and a 1,4-relationship between the base moiety and the hydroxymethyl group.

[101] Kozlov I. A., Zielinski M., Allart B., Kerremans L., Van Aerschot A., Busson R., Herdewijn P. and Orgel L. E. (2000) Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides. Chemistry. 6, 151-155. [102] Herdewijn P., Allart B., De Bouvere B., De Winter H., Hendrix C., Hossain N., Schepers G., Verheggen I., Wroblowski B. and Van Aerschot A. (1999) Properties of oligonucleotides with six membered base moiety and the hydroxymethyl group mimics and a 1,4-relationship between the base moiety and the hydroxymethyl group.

[103] Fisher M., Abramov M., Van Aerschot A., Xu D., Juliano R. L. and Herdewijn P. (2007) Inhibition of MDR1 expression with altritol-modified siRNAs. Nucleic Acids Res. 35,

[104] Kenski D. M., Cooper A. J., Li J. J., Willingham A. T., Haringsma H. J., Young T. A., Kuklin N. A., Jones J. J., Cancilla M. T., McMasters D. R., Mathur M., Sachs A. B. and Flanagan W. M. (2010) Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and

[105] Langkjaer N., Pasternak A. and Wengel J. (2009) UNA (unlocked nucleic acid): a flexible RNA mimic that allows engineering of nucleic acid duplex stability. Bioorg Med

[106] Kenski D. M., Cooper A. J., Li J. J., Willingham A. T., Haringsma H. J., Young T. A., Kuklin N. A., Jones J. J., Cancilla M. T., McMasters D. R., Mathur M., Sachs A. B. and Flanagan W. M. (2010) Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and

[107] Werk D., Wengel J., Wengel S. L., Grunert H. P., Zeichhardt H. and Kurreck J. (2010) Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to

[108] Brown S. C., Thomson S. A., Veal J. M. and Davis D. G. (1994) NMR solution structure

[109] Demidov V. V., Potaman V. N., Frank-Kamenetskii M. D., Egholm M., Buchard O., Sonnichsen S. H. and Nielsen P. E. (1994) Stability of peptide nucleic acids in human

[110] Potenza N., Moggio L., Milano G., Salvatore V., Di Blasio B., Russo A. and Messere A. (2008) RNA interference in mammalia cells by RNA-3'-PNA chimeras. Int J Mol Sci. 9,

[111] Harborth J., Elbashir S. M., Vandenburgh K., Manninga H., Scaringe S. A., Weber K. and Tuschl T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense

[112] Hall A. H., Wan J., Shaughnessy E. E., Ramsay Shaw B. and Alexander K. A. (2004) RNA interference using boranophosphate siRNAs: structure-activity relationships.

inhibit the heart-pathogenic coxsackievirus B3. FEBS Lett. 584, 591-598.

of a peptide nucleic acid complexed with RNA. Science. 265, 777-780.

serum and cellular extracts. Biochem Pharmacol. 48, 1310-1313.

Nucleosides & Nucleotides. 18, 1371-1376.

Nucleosides & Nucleotides. 18, 1371-1376.

in vivo. Nucleic Acids Res. 38, 660-671.

in vivo. Nucleic Acids Res. 38, 660-671.

Nucleic Acid Drug Dev. 13, 83-105.

Nucleic Acids Res. 32, 5991-6000.

1064-1074.

299-315.

Chem. 17, 5420-5425.


and a 1,4-relationship between the base moiety and the hydroxymethyl group. Nucleosides & Nucleotides. 18, 1371-1376.

[101] Kozlov I. A., Zielinski M., Allart B., Kerremans L., Van Aerschot A., Busson R., Herdewijn P. and Orgel L. E. (2000) Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides. Chemistry. 6, 151-155.

222 Practical Applications in Biomedical Engineering

Biol. 5, 343-355.

Res. 31, 3185-3193.

439-447.

Nucleic Acids Res. 15, 6131-6148.

mammalian cells. FEBS Lett. 579, 3115-3118.

with 4'-thioribonucleosides. Chembiochem. 8, 2133-2138.

recognition of DNA and RNA. Chem Biol. 8, 1-7.

[88] Bramsen J. B., Laursen M. B., Nielsen A. F., Hansen T. B., Bus C., Langkjaer N., Babu B. R., Hojland T., Abramov M., Van Aerschot A., Odadzic D., Smicius R., Haas J., Andree C., Barman J., Wenska M., Srivastava P., Zhou C., Honcharenko D., Hess S., Muller E., Bobkov G. V., Mikhailov S. N., Fava E., Meyer T. F., Chattopadhyaya J., Zerial M., Engels J. W., Herdewijn P., Wengel J. and Kjems J. (2009) A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high

[89] De Mesmaeker A., Altmann K. H., Waldner A. and Wendeborn S. (1995) Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr Opin Struct

[90] Inoue H., Hayase Y., Imura A., Iwai S., Miura K. and Ohtsuka E. (1987) Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides.

[91] Dande P., Prakash T. P., Sioufi N., Gaus H., Jarres R., Berdeja A., Swayze E. E., Griffey R. H. and Bhat B. (2006) Improving RNA interference in mammalian cells by 4'-thiomodified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications. J Med Chem. 49, 1624-1634. [92] Hoshika S., Minakawa N., Kamiya H., Harashima H. and Matsuda A. (2005) RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured

[93] Hoshika S., Minakawa N., Shionoya A., Imada K., Ogawa N. and Matsuda A. (2007) Study of modification pattern-RNAi activity relationships by using siRNAs modified

[94] Braasch D. A. and Corey D. R. (2001) Locked nucleic acid (LNA): fine-tuning the

[95] Grunweller A., Wyszko E., Bieber B., Jahnel R., Erdmann V. A. and Kurreck J. (2003) Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids

[96] Elmen J., Thonberg H., Ljungberg K., Frieden M., Westergaard M., Xu Y., Wahren B., Liang Z., Orum H., Koch T. and Wahlestedt C. (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res. 33,

[97] Hornung V., Guenthner-Biller M., Bourquin C., Ablasser A., Schlee M., Uematsu S., Noronha A., Manoharan M., Akira S., de Fougerolles A., Endres S. and Hartmann G. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in

[98] Mook O. R., Baas F., de Wissel M. B. and Fluiter K. (2007) Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo. Mol Cancer Ther. 6, 833-843. [99] Bramsen J. B., Laursen M. B., Damgaard C. K., Lena S. W., Babu B. R., Wengel J. and Kjems J. (2007) Improved silencing properties using small internally segmented

[100] Herdewijn P., Allart B., De Bouvere B., De Winter H., Hendrix C., Hossain N., Schepers G., Verheggen I., Wroblowski B. and Van Aerschot A. (1999) Properties of oligonucleotides with six membered base moiety and the hydroxymethyl group mimics

plasmacytoid dendritic cells through TLR7. Nat Med. 11, 263-270.

interfering RNAs. Nucleic Acids Res. 35, 5886-5897.

stability and low toxicity. Nucleic Acids Res. 37, 2867-2881.


[113] Schwarz D. S., Tomari Y. and Zamore P. D. (2004) The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr Biol. 14, 787-791.

Structure - Functions Relations in Small Interfering RNAs 225

using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem. 18, 1450-1459. [128] Muratovska A. and Eccles M. R. (2004) Conjugate for efficient delivery of short

[129] McNamara J. O., 2nd, Andrechek E. R., Wang Y., Viles K. D., Rempel R. E., Gilboa E., Sullenger B. A. and Giangrande P. H. (2006) Cell type-specific delivery of siRNAs with

[130] Chiu Y. L. and Rana T. M. (2002) RNAi in human cells: basic structural and functional

[131] Martinez J., Patkaniowska A., Urlaub H., Luhrmann R. and Tuschl T. (2002) Singlestranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 110, 563-574. [132] De Paula D., Bentley M. V. and Mahato R. I. (2007) Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting. Rna. 13, 431-456. [133] Gaglione M. and Messere A. (2010) Recent progress in chemically modified siRNAs.

[134] Burnett J. R. and Barrett P. H. (2002) Apolipoprotein B metabolism: tracer kinetics,

[135] Feinberg E. H. and Hunter C. P. (2003) Transport of dsRNA into cells by the

[136] Potocky T. B., Menon A. K. and Gellman S. H. (2003) Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa

[137] Richard J. P., Melikov K., Vives E., Ramos C., Verbeure B., Gait M. J., Chernomordik L. V. and Lebleu B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of

[138] Poehlmann T. G., Ruediger T., Schaefer H., Koehn S., Imhof D., Seyfarth L., Schubert U. S. and Markert U. R. (2009) Development of small interfering RNA selectively activated in target cells. In RNAi2009: ncRNA: Bridging Biology and Therapy (Media L.

[139] Hicke B. J. and Stephens A. W. (2000) Escort aptamers: a delivery service for diagnosis

[140] Dassie J. P., Liu X. Y., Thomas G. S., Whitaker R. M., Thiel K. W., Stockdale K. R., Meyerholz D. K., McCaffrey A. P., McNamara J. O., 2nd and Giangrande P. H. (2009) Systemic administration of optimized aptamer-siRNA chimeras promotes regression of

[141] Xia C. F., Boado R. J. and Pardridge W. M. (2009) Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology. Mol Pharm. 6,

[142] Xia C. F., Zhang Y., Boado R. J. and Pardridge W. M. (2007) Intravenous siRNA of brain cancer with receptor targeting and avidin-biotin technology. Pharm Res. 24, 2309-

[143] Xia W. and Low P. S. Folate-targeted therapies for cancer. (2010) J Med Chem. 53,

[144] Kim S. H., Mok H., Jeong J. H., Kim S. W. and Park T. G. (2006) Comparative evaluation of target-specific GFP gene silencing efficiencies for antisense ODN,

interfering RNA (siRNA) into mammalian cells. FEBS Lett. 558, 63-68.

aptamer-siRNA chimeras. Nat Biotechnol. 24, 1005-1015.

features of small interfering RNA. Mol Cell. 10, 549-561.

models, and metabolic studies. Crit Rev Clin Lab Sci. 39, 89-137.

P., ed.)^eds.). pp. 16-17, Library Publishing Media, Oxford, UK.

PSMA-expressing tumors. Nat Biotechnol. 27, 839-849.

transmembrane protein SID-1. Science. 301, 1545-1547.

Mini Rev Med Chem. 10, 578-595.

cells. J Biol Chem. 278, 50188-50194.

cellular uptake. J Biol Chem. 278, 585-590.

and therapy. J Clin Invest. 106, 923-928.

747-751.

2316.

6811-6824.


using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem. 18, 1450-1459.

[128] Muratovska A. and Eccles M. R. (2004) Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells. FEBS Lett. 558, 63-68.

224 Practical Applications in Biomedical Engineering

Lett. 16, 3238-3240.

Biotechnol. 23, 1002-1007.

Cell. 10, 537-548.

[113] Schwarz D. S., Tomari Y. and Zamore P. D. (2004) The RNA-induced silencing

[114] Lee M., Simon A. D., Stein C. A. and Rabbani L. E. (1999) Antisense strategies to

[115] Prakash T. P., Kraynack B., Baker B. F., Swayze E. E. and Bhat B. (2006) RNA interference by 2',5'-linked nucleic acid duplexes in mammalian cells. Bioorg Med Chem

[116] Iwase R., Toyama T. and Nishimori K. (2007) Solid-phase synthesis of modified RNAs containing amide-linked oligoribonucleosides at their 3'-end and their application to

[117] Saenger W. (1984) Forces stabilizing association between bases: Hydrogen bonding and base stacking. In Principle of nucleic acids structure. Springer-Verlag, New York. [118] Agris P. F., Sierzputowska-Gracz H., Smith W., Malkiewicz A., Sochacka E. and Nawrot B. (1992) Thiolation of uridine carbon-2 restricts the motional dynamics of the

[119] Davis D. R., Veltri C. A. and Nielsen L. (1998) An RNA model system for investigation of pseudouridine stabilization of the codon-anticodon interaction in tRNALys,

[120] Sipa K., Sochacka E., Kazmierczak-Baranska J., Maszewska M., Janicka M., Nowak G. and Nawrot B. (2007) Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA. RNA. 13, 1301-1316. [121] Morrissey D. V., Lockridge J. A., Shaw L., Blanchard K., Jensen K., Breen W., Hartsough K., Machemer L., Radka S., Jadhav V., Vaish N., Zinnen S., Vargeese C., Bowman K., Shaffer C. S., Jeffs L. B., Judge A., MacLachlan I. and Polisky B. (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat

[122] Schwarz D. S., Hutvagner G., Haley B. and Zamore P. D. (2002) Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol

[123] Chen P. Y., Weinmann L., Gaidatzis D., Pei Y., Zavolan M., Tuschl T. and Meister G. (2008) Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand

[124] Lorenz C., Hadwiger P., John M., Vornlocher H. P. and Unverzagt C. (2004) Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver

[125] Wolfrum C., Shi S., Jayaprakash K. N., Jayaraman M., Wang G., Pandey R. K., Rajeev K. G., Nakayama T., Charrise K., Ndungo E. M., Zimmermann T., Koteliansky V., Manoharan M. and Stoffel M. (2007) Mechanisms and optimization of in vivo delivery

[126] Thomas M., Kularatne S. A., Qi L., Kleindl P., Leamon C. P., Hansen M. J. and Low P. S. (2009) Ligand-targeted delivery of small interfering RNAs to malignant cells and

[127] Moschos S. A., Jones S. W., Perry M. M., Williams A. E., Erjefalt J. S., Turner J. J., Barnes P. J., Sproat B. S., Gait M. J. and Lindsay M. A. (2007) Lung delivery studies

transfer RNA wobble position nucleoside. J. Am. Chem. Soc. 114, 2652-2656.

complex is a Mg2+-dependent endonuclease. Curr Biol. 14, 787-791.

inhibit restenosis. Antisense Nucleic Acid Drug Dev. 9, 487-492.

siRNA. Nucleosides Nucleotides Nucleic Acids. 26, 1451-1454.

tRNAHis and tRNATyr. J Biomol Struct Dyn. 15, 1121-1132.

selection and targeting specificity. RNA. 14, 263-274.

of lipophilic siRNAs. Nat Biotechnol. 25, 1149-1157.

cells. Bioorg Med Chem Lett. 14, 4975-4977.

tissues. Ann N Y Acad Sci. 1175, 32-39.


synthetic siRNA, and siRNA plasmid complexed with PEI-PEG-FOL conjugate. Bioconjug Chem. 17, 241-244.

Structure - Functions Relations in Small Interfering RNAs 227

[160] Kubo T., Zhelev Z., Ohba H. and Bakalova R. (2007) Modified 27-nt dsRNAs with dramatically enhanced stability in serum and long-term RNAi activity.

[161] Rose S. D., Kim D. H., Amarzguioui M., Heidel J. D., Collingwood M. A., Davis M. E., Rossi J. J. and Behlke M. A. (2005) Functional polarity is introduced by Dicer processing

[162] Sano M., Sierant M., Miyagishi M., Nakanishi M., Takagi Y. and Sutou S. (2008) Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference

[163] Masquida B. and Westhof E. (2000) On the wobble GoU and related pairs. RNA. 6, 9-

[164] Ding H., Liao G., Wang H. and Zhou Y. (2007) Asymmetrically designed siRNAs and shRNAs enhance the strand specificity and efficacy in RNAi. J RNAi Gene Silencing. 4,

[165] Patzel V., Rutz S., Dietrich I., Koberle C., Scheffold A. and Kaufmann S. H. (2005) Design of siRNAs producing unstructured guide-RNAs results in improved RNA

[166] Hu X., Hipolito S., Lynn R., Abraham V., Ramos S. and Wong-Staal F. (2004) Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense

[169] Marques J. T., Devosse T., Wang D., Zamanian-Daryoush M., Serbinowski P., Hartmann R., Fujita T., Behlke M. A. and Williams B. R. (2006) A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.

[170] Guo S. and Kemphues K. J. (1995) par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed.

[171] Tijsterman M., Ketting R. F., Okihara K. L., Sijen T. and Plasterk R. H. (2002) RNA helicase MUT-14-dependent gene silencing triggered in C. elegans by short antisense

[172] Amarzguioui M., Rossi J. J. and Kim D. (2005) Approaches for chemically synthesized

[173] Collingwood M. A., Rose S. D., Huang L., Hillier C., Amarzguioui M., Wiiger M. T., Soifer H. S., Rossi J. J. and Behlke M. A. (2008) Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs. Oligonucleotides.

[174] Kim D. H., Behlke M. A., Rose S. D., Chang M. S., Choi S. and Rossi J. J. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol.

[175] Reynolds A., Anderson E. M., Vermeulen A., Fedorov Y., Robinson K., Leake D., Karpilow J., Marshall W. S. and Khvorova A. (2006) Induction of the interferon response

by siRNA is cell type- and duplex length-dependent. RNA. 12, 988-993.

transcripts from the same genetic locus. Nucleic Acids Res. 32, 4609-4617. [167] Patzel V. (2007) In silico selection of active siRNA. Drug Discov Today. 12, 139-148. [168] Lingel A., Simon B., Izaurralde E. and Sattler M. (2004) Nucleic acid 3'-end recognition

by the Argonaute2 PAZ domain. Nat Struct Mol Biol. 11, 576-577.

siRNA and vector-mediated RNAi. FEBS Lett. 579, 5974-5981.

of short substrate RNAs. Nucleic Acids Res. 33, 4140-4156.

interference efficiency. Nat Biotechnol. 23, 1440-1444.

activity and strand selection. Nucleic Acids Res. 36, 5812-5821.

Oligonucleotides. 17, 445-464.

Nat Biotechnol. 24, 559-565.

RNAs. Science. 295, 694-697.

Cell. 81, 611-620.

18, 187-200.

23, 222-226.

15.

269-280.


[160] Kubo T., Zhelev Z., Ohba H. and Bakalova R. (2007) Modified 27-nt dsRNAs with dramatically enhanced stability in serum and long-term RNAi activity. Oligonucleotides. 17, 445-464.

226 Practical Applications in Biomedical Engineering

Bioconjug Chem. 17, 241-244.

Bioconjug Chem. 20, 5-14.

Delivery. Drugs Future. 34, 721.

host gene. Cell Host Microbe. 5, 84-94.

interference? J Clin Invest. 117, 3615-3622.

Biochem Biophys Res Commun. 329, 516-521.

Bioconjug Chem. 20, 5-14.

12, 1197-1205.

Science. 311, 195-198.

Biotechnol. 21, 324-328.

Lett. 557, 193-198.

mammalian cell culture. Chem Biol Drug Des. 70, 113-122.

14, 577-583.

443-453.

synthetic siRNA, and siRNA plasmid complexed with PEI-PEG-FOL conjugate.

[145] Zhang K., Wang Q., Xie Y., Mor G., Sega E., Low P. S. and Huang Y. (2008) Receptormediated delivery of siRNAs by tethered nucleic acid base-paired interactions. RNA.

[146] Jeong J. H., Mok H., Oh Y. K. and Park T. G. (2009) siRNA conjugate delivery systems.

[147] Kawakami S. and Hashida M. (2007) Targeted delivery systems of small interfering

[148] Leng Q., Woodle M. C., Lu P. Y. and Mixson A. J. (2009) Advances in Systemic siRNA

[149] Lv H., Zhang S., Wang B., Cui S. and Yan J. (2006) Toxicity of cationic lipids and cationic polymers in gene d Xia W. and Low P. S. Folate-targeted therapies for cancer.

[150] de Fougerolles A., Vornlocher H. P., Maraganore J. and Lieberman J. (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov. 6,

[151] Wu Y., Navarro F., Lal A., Basar E., Pandey R. K., Manoharan M., Feng Y., Lee S. J., Lieberman J. and Palliser D. (2009) Durable protection from Herpes Simplex Virus-2 transmission following intravaginal application of siRNAs targeting both a viral and

[152] Blidner R. A., Hammer R. P., Lopez M. J., Robinson S. O. and Monroe W. T. (2007) Fully 2'-deoxy-2'-fluoro substituted nucleic acids induce RNA interference in

[153] Corey D. R. (2007) Chemical modification: the key to clinical application of RNA

[154] Jeong J. H., Mok H., Oh Y. K. and Park T. G. (2009) siRNA conjugate delivery systems.

[155] Jackson A. L., Burchard J., Leake D., Reynolds A., Schelter J., Guo J., Johnson J. M., Lim L., Karpilow J., Nichols K., Marshall W., Khvorova A. and Linsley P. S. (2006) Positionspecific chemical modification of siRNAs reduces "off-target" transcript silencing. RNA.

[156] Macrae I. J., Zhou K., Li F., Repic A., Brooks A. N., Cande W. Z., Adams P. D. and Doudna J. A. (2006) Structural basis for double-stranded RNA processing by Dicer.

[157] Myers J. W., Jones J. T., Meyer T. and Ferrell J. E., Jr. (2003) Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat

[158] Hohjoh H. (2004) Enhancement of RNAi activity by improved siRNA duplexes. FEBS

[159] Ohnishi Y., Tokunaga K. and Hohjoh H. (2005) Influence of assembly of siRNA elements into RNA-induced silencing complex by вилко-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element.

RNA by systemic administration. Drug Metab Pharmacokinet. 22, 142-151.

(2010) J Med Chem. 53, 6811-6824.elivery. J Control Release. 114, 100-109.


[176] Bridge A. J., Pebernard S., Ducraux A., Nicoulaz A. L. and Iggo R. (2003) Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet. 34, 263-264.

**Chapter 9** 

© 2012 de Campos-Takaki et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 de Campos-Takaki et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Microbiological Chitosan:** 

and Galba Maria de Campos-Takaki

http://dx.doi.org/10.5772/54453

**1. Introduction** 

some contribution [6, 7].

Additional information is available at the end of the chapter

**Potential Application as Anticariogenic Agent** 

Thayza Christina Montenegro Stamford, Thatiana Montenegro Stamford-Arnaud, Horacinna Maria de Medeiros Cavalcante, Rui Oliveira Macedo

Dental caries is the most prevalent oral disease that affects a significant part of the world population, especially in less developed countries. It is universally accepted that the dental caries is a chronic and multifactorial disease [1-3]. The permanence of the bacterial plaque on the tooth surface will lead to loss of minerals constituents of the dental enamel promoting the installation of the carie disease. The carious lesion is characterized by the tooth structure (hydroxyapatite) demineralization by the production of organic acids, such as lactic acid, resulting from bacterial (dental biofilm) metabolism. This results in loss of calcium and phosphate ions which subsequently diffuse out of the tooth. In this complex process, the microorganisms, particularly *Streptococcus* species, have an important role in its etiology [3-5].

Many oral *Streptococcus* in the presence of carbohydrate produce organics acids and insoluble glucans which serve as binding sites for bacteria on the tooth surface, forming the biofilm. The sucrose plays an important role in carie development, influencing on biofilm acidogenicity and cariogenic microflora. The high cariogenicity of dental plaque formed in the presence of sucrose can be mainly explained by the high concentration of insoluble glucans on its matrix, the low inorganic concentration and its protein composition may have

Specifics microorganisms are associated with dental plaque formation, development and maturation. The *Streptococcus mutans* is a member of the oral microbial community which plays a key role in formation of cariogenic biofilms. The key factors of *S. mutans* cariogenic are the production of a great variety of carbohydrates, which generate low pH and cause the

consequent demineralization of the tooth enamel [3, 8-10].

